WO1999067427A1 - Means and methods for monitoring nucleoside reverse transcriptase inhibitor antiretroviral therapy and guiding therapeutic decisions in the treatment of hiv/aids - Google Patents
Means and methods for monitoring nucleoside reverse transcriptase inhibitor antiretroviral therapy and guiding therapeutic decisions in the treatment of hiv/aids Download PDFInfo
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- WO1999067427A1 WO1999067427A1 PCT/US1999/014486 US9914486W WO9967427A1 WO 1999067427 A1 WO1999067427 A1 WO 1999067427A1 US 9914486 W US9914486 W US 9914486W WO 9967427 A1 WO9967427 A1 WO 9967427A1
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- C12Q1/703—Viruses associated with AIDS
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- 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 whose infection has become resistant to a particular antiretroviral drug regimen.
- This invention also relates to the screening of candidate antiretroviral drugs for their capacity to inhibit viruses, selected viral sequences and/or viral proteins. More particularly, this invention relates to the determination of nucleoside reverse transcriptase inhibitor resistance using phenotypic susceptibility tests and/or genotypic tests.
- 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 change in shape, size or charge of the active site, substrate binding site or surrounding regions of the enzyme. Mutants resistant to antiretroviral agents have been detected at low levels before the initiation of therapy. (Mohri H, Singh MK,
- the selective pressure of antiretroviral therapy provides these drug-resistant mutants with a competitive advantage and thus they come to represent the dominant quasispecies (Frost SDW, McLean AR (1994) AIDS 8, 323-332) (Kellam P, Boucher CAB, Tijnagal JMGH (1994) J Gen Virol 75, 341-351) ultimately leading to drug resistance and virologic failure in the patient.
- zidovudine ZVD: Retrovir, Glaxo Wellcome, Uxbridge, UK
- zalcitabine ddC: HIVID, Hoffman-
- nevirapme (Viramune, Boeh ⁇ nger Ingelheim, Ingelheim am Rhein, Germany) and delavirdme (Resc ⁇ ptor, Pharmacia & Upjohn, Kalamazoo, MI, USA) and efavirenz (EPV, ) are licensed m the USA. All these agents have demonstrated at least short-term antiviral activity and, therefore, it is not surprising that, as they exert a selective pressure on HIV, drug-resistant mutants arise during therapy. Whilst these drugs are normally used m combination regimens, many of the available resistance data arise from phase I/II monotherapy studies. Mutations observed during monotherapy may not accurately reflect mutations responsible for resistance that develops m the presence of pressure from several agents acting at the same site and, hence, on the same gene.
- RTIs reverse transcriptase inhibitors
- Nucleotide sequencing of HIV RT has revealed a number of mutations which can influence viral sensitivity to ZDV and which may be used as genotypic markers for the presence of ZDV resistance (Kellam P, Boucher CAB, Tijnagal JMGH et al. (1994) J Gen Virol 75, 341-351) (Boucher CAB, Tersmette M, Lange JMA, et al . (1990) Lancet 336, 585-590) (Lopez-Galindez C, Rojas JM, Najera R, et al . (1991) PNAS 88, 4280-4284) .
- a range of mutants with increasing levels of resistance appear in an ordered manner, with the sequential appearance of these mutations being associated with incremental reductions in viral sensitivity to ZDV
- Virus with additional mutations may then appear, most commonly a substitution at codon 41 (Met41-Leu) , followed by further additional mutations at codons 67, (Asp67-Asn) and 219 (Lys219 ⁇ Gln) or the reappearance of the codon 70 mutation ⁇ id) .
- Viral strains resistant to ZDV exhibit cross-resistance to other nucleoside analogues containing the 3'-azido group such as 3' -azido-2' , 3' -dideoxyuridine (AZU) (Rooke R, Parniak MA, Tremblay M, et al . (1991) Antimi crob Agents Chermother 35, 988-991).
- Cross-resistance to stavudine a thymidine-based analogue which lacks a 3'-azido moiety, has also been reported by one group in both a laboratory strain of HIV and one of 11 clinical isolates (ibid) .
- Resistance to ddl is mediated through a Leu74-Val mutation which produces a six-fold to 26-fold reduction in sensitivity, but may partially restore susceptibility to ZDV in vitro by antagonism of the codon 215 mutation.
- This mutation also reduces sensitivity to ddC by around ten-fold (id) .
- the frequency of the codon 74 mutation has been reported to have increased from zero at the start of therapy to 56% at week 24 in a group of 64 persons with a mean baseline CD4 cell count of 129/mm who switched to ddl having previously received ZDV (Kozal MJ, Kroodsma K,
- Virus with a mutation at codon 65 has been isolated from several patients receiving long-term treatment with ddl or ddC. This is associated with a three-fold to five-fold increase in the IC 50 of ddl with a five-fold to ten-fold reduction in ddC sensitivity and a 20-fold reduction in susceptibility to 3TC (id) (Gu Z, Gao Q, Fang H, et al . (1994) Antimicrob Agents Chemother 38, 275-281.
- a mutation at codon 69 (Thr69-Asp) , which leads to a five-fold reduction in sensitivity to ddC but does not appear to result in cross-resistance to other nucleoside analogues, is the most frequent mutation selected for by ddC in vivo (id) (Fitzgibbon JE, Howe ⁇ l RM, Haberzettl CA, et al . (1992) Antimicrob Agents Chemother 36, 153-157) . The development of resistance to ddC has recently been reviewed elsewhere (Craig C, Moyle G (1997) AIDS 11, 271-279) .
- Combination therapy with ZDV/ddC or ZDV/ddl may influence the rate of emergence of resistance and may suppress some of the mutations observed during monotherapy but may result in the appearance of novel (and hence possibly more compromised) mutational patterns.
- Novel mutation patterns may emerge during combination therapy. Isolates with a unique pattern of amino acid substitutions at codons 62, 75, 77, 116, and 151 have occasionally been identified in patients receiving prolonged combination therapy with ZDV plus ddl or alternating ZDV/ddC: these are resistant to both drugs (id) (Shafer RW, Kozal MJ, Winters MA, et al . (1994) J Infect Di sease 169, 722-729) (Shirasaka T, Kavlick MF, Ueno T, et al . (1995) PNAS 92, 2398-2402) and confer cross-resistance to stavudine and partial cross-resistance to 3TC.
- vi tro selection -of HIV resistant to d4T confirmed by site-directed mutagenesis, has identified a mutation at codon 75 (Val75-Thr) which confers a seven-fold increase in IC 5C , as well as reduced susceptibility to both ddl and ddC (Lacey SF, Larder BA (1994) Antimicrob Agent Chemother 38, 1428-1432) .
- a mutation at codon 50 leading to a 30-fold reduction in d4T sensitivity, but which does not appear to confer cross-resistance to other nucleoside analogues has also been observed in vi tro (Gu Z, Gao Q, Fank H, et al . (1994) Leukemia 8, Suppl . 1, 166-169).
- Choice of initial and subsequent therapy for HIV infection should be uncompromising in terms of activity but also planned and based rationally on knowledge of resistance and cross-resistance patterns to maintain a wide base of future therapy options.
- Still another object of this invention is to provide a test and methods for evaluating the biological effectiveness of candidate drug compounds that act on specific viruses, viral genes and/or viral proteins particularly with respect to viral drug resistance associated with nucleoside reverse transcriptase inhibitors. It is also an object of this invention to provide the means and compositions for evaluating HIV antiretroviral drug resistance and susceptibility. This and other objects of this invention will be apparent from the specification as a whole.
- the present invention relates to methods of monitoring, using phenotypic and genotypic methods, the clinical progression of human immunodeficiency virus infection and its response to antiviral therapy.
- the invention is also based, m part, on the discovery that genetic changes m HIV reverse transcriptase (RT) which confer resistance to antiretroviral therapy may be rapidly determined directly from patient plasma HIV RNA using phenotypic or genotypic methods.
- the methods utilize nucleic acid amplification based assays, such as polymerase chain reaction.
- PCR based assays will be referred to as PCR based assays.
- T69SSS may also be associated with mutations associated with resistance to AZT (e.g. M41L, L210W, T215Y) and 3TC
- ddl/ddC L74V
- m NRTI-susceptibility including a decrease d4T susceptibility and a decrease in susceptibility to AZT, ddC, ddl, 3TC and abacavir.
- mutation/insertion at codon 69 in RT may be associated with mutations associated with resistance to multi-NRTIs at codon 62 (e.g. A62V) and/or a novel mutation at codon 75 (e.g. V75M) .
- mutations specifically associated with resistance to: AZT at codons 41, 67, 210, 215 and 219 e.g. M41L, D67N, L210W, T215Y, K219Q
- T215V novel mutation at codon 215
- This invention is based in part on the discovery of four or more mutations associated with AZT resistance selected from the group consisting of codons 41, 67, 70, 210, 215 and/or 219 (e.g. M41L, D67N, K70R, L210W, T215Y/F, K219Q) either alone or in combination with , a mutation at codon 74 (associated with ddl resistance -
- V75M, V75S and/or 219 (K219N) of HIV reverse transcriptase in nucleoside reverse transcriptase inhibitor treated patient (s) in which the presence of the mutations correlate with a decrease in d4T susceptibility.
- the mutations were found in plasma HIV RNA after a period of time following initiation of NRTI therapy. It was observed through the construction, by site directed mutagenesis, of resistance test vectors containing the single site mutation at codon 75 (V75I) did not alter d4T susceptibility but increased AZT susceptibility. It was also observed using site directed mutagenesis that the single site mutation at codon 151 (Q151M) reduced d4T and AZT susceptibility.
- A62V + V75I had no effect on d4T susceptibility. It was also found that the A62V mutation did not alter the reduced AZT susceptibility caused by the V75I mutation. In yet further studies using site directed mutagenesis, it was observed that a combination of three mutations at codons 62, 69 and 75 (e.g. A62V + T69SSA + V75I) did not reduce d4T susceptibility more than the T69SSA mutation alone. It was also observed in the case of the combination of three mutations at codons 62, 69 and 75 (e.g.
- M41L + A62V + T69SSA + T215Y did reduce d4T susceptibility more than the T69SSA mutation alone or the T69SSA + A62V double mutant. It was also observed in the case of the combination of four mutations at codons 41, 62, 69 and 215 (e.g. M41L + A62V + T69SSA + T215Y) that the combination of all four mutations reduced AZT susceptibility more than the combination of M41L and T215Y mutations alone. In yet further studies using site directed mutagenesis, it was observed that a combination of five mutations at codons 41, 62, 69, 184 and 215 (e.g.
- M41L + A62V + T69SSA + M184V + T215Y did reduce d4T susceptibility more than the T69SSA mutation alone or the A62V + T69SSA double mutant. It was also observed in the case of the combination of five mutations at codons 41, 62, 69, 184 and 215 (e.g. M41L + A62V + T69SSA + M184V + T215Y) that the M184V mutation suppressed the reduced AZT susceptibility caused by the combination of M41L, A62V, T69SSA and T215Y mutations.
- T69SSA - SSA69T T69SSA - SSA69T
- Reversion of the T69SSA mutation reduced d4T resistance (i.e. increased susceptibility) and also reduced AZT resistance (i.e. increased susceptibility).
- PCR based assays including phenotypic and genotypic assays, may be used to detect mutations at codon 69 in combination with mutations at other codons including 41 and/or 215 of HIV RT which correlate with a specific pattern of resistance to antiretroviral therapies and subsequent immunologic decline.
- PCR based assays may be used to detect mutations at codon 69 (T69SSA, T69SSG, T69SSS) in combination with mutations at other codons including 41 (M41L) , 210 (L210W) , 215 (T215Y), 184 (M184V) and/or 74 (L74V) of HIV RT which correlate, as described herein, with a specific pattern of resistance to antiretroviral therapies and subsequent immunologic decline.
- PCR based assays may be used to detect mutations at codon 62, 75, 77, 116 or 151 either alone or in combination with mutations at other codons including 41, 67, 210, 215, 219, 184, 69 and/or 215 of HIV RT which correlate, as described herein, with resistance to antiretroviral therapies and immunologic decline.
- mutations at the aforementioned codons include, but are not limited to
- PCR based assays including phenotypic and genotypic assays, may be used to detect four or more mutations in RT at codons in the group consisting of 41, 67, 70, 210, 215 and/or 219 (e.g.
- PCR based assays including phenotypic and genotypic assays, may be used to detect mutations at codon 69 in combination with mutations at other codons including 41 and/or 215 of HIV RT which correlate with a specific pattern of resistance to antiretroviral therapies and subsequent immunologic decline.
- PCR based assays including phenotypic and genotypic assays, may be used to detect mutations at codon 69 in combination with mutations at other codons including 41, 210, 215, 184 and/or 74 of HIV RT which correlate with a specific pattern of resistance to antiretroviral therapies and subsequent immunologic decline.
- PCR based assays may be used to detect mutations at codon 69 m combination with mutations at other codons including 62 and/or 75 of HIV RT which correlate with a specific pattern of resistance to antiretroviral therapies and subsequent immunologic decline.
- PCR based assays including phenotypic and genotypic assays, may be used to detect mutations at codon 62, 75, 77, 116 and 151 either alone or combination with mutations at other codons including 41, 67, 210, 215, 219, 184, 69 and/or T215V of HIV RT which correlate with a specific pattern of resistance to antiretroviral therapies and subsequent immunologic decline.
- the timing at which a modification of the therapeutic regimen should be made, following the assessment of the antiretroviral therapy using PCR based assays, may depend on several factors including the patient's viral load, CD4 count, and prior treatment history.
- a method for assessing the effectiveness of a nucleoside reverse transcriptase antiretroviral drug comprising: (a) introducing a resistance test vector comprising a patient- derived segment and an indicator gene into a host cell; (b) cultu ⁇ ng the host cell from step (a) ; (c) measuring expression of the indicator gene m 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 m the absence of the NRTI anti-HIV drug, wherein a test concentration of the NRTI, 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 non-nucleoside reverse transcriptase antiretroviral therapy in a patient comprising: (a) developing a standard curve of drug susceptibility for an NRTI anti-HIV drug; (b) determining NRTI anti-HIV drug susceptibility in the patient using the susceptibility test described above; and (c) comparing the NRTI anti-HIV drug susceptibility in step (b) with the standard curve determined in step (a) , wherein a decrease in NRTI anti- HIV susceptibility indicates development of anti-HIV drug resistance in the patient.
- This invention also provides a method for evaluating the biological effectiveness of a candidate 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 anti-viral drug compound, wherein a test concentration of the candidate 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 patient-derived segment 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.
- 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 RT gene and an indicator gene.
- 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 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.
- the amount of luciferase is determined and the percentage (%) inhibition is calculated at the different drug concentrations tested.
- phenotypic drug susceptibility profiles Data are analyzed by plotting the percent inhibition of luciferase activity vs. log 10 concentration. This plot is used to calculate the drug concentration that is required to inhibit virus replication by 50% (IC 50 ) or by 95% (IC 95 ) . 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 (delavirdine) , and a protease inhibitor (ritonavir) are shown.
- AZA nucleoside reverse transcriptase inhibitor
- delavirdine non-nucleoside reverse transcriptase inhibitor
- ritonavir 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 baseli ⁇ e (pre-treatment) sample or a drug susceptible virus control, such as PNL4-3 or HXB-2, when a baseline sample is not available.
- FIG. 4 d4T Resistant Patient Isolates Multi-NRTI Resistance Mutations. These four viruses exhibit reduced susceptibility to d4T (4-12 fold) and contain RT mutations associated with multi-NRTI resistance (A62V, V75I, F77L, F116Y, Q151M) . Some viruses also contain mutations specifically associated with resistance to AZT (M41L, D67N, L210W, T215Y, K219Q) , 3TC (M184V/I), or ddC (T69D), or a previously undescribed mutation (T215V) . Mutations for the test virus are listed below the test virus profile. Mutations within parentheses indicate that the virus population was comprised of a mixture of wildtype and mutant. Genotype described here is partial. For a complete description of the patient genotype, see example 3.
- FIG. 5 d4T Resistant Patient Isolates: T69SSX Mutations / Insertions. These four viruses exhibit reduced susceptibility to d4T (2-10 fold) and contain previously undescribed mutations/insertions in RT (T69SSA, T69SSG, T69SSS) . These viruses also contain mutations associated with resistance to AZT (M41L, L210W, T215Y) and 3TC
- viruses also contain a mutation associated with resistance to multi-NRTIs (A62V) and/or a previously undescribed mutation (V75M) . Mutations for the test virus are listed below the test virus profile. Mutations within parentheses indicate that the virus population was comprised of a mixture of wildtype and mutant. Genotype described here is partial. For a complete description of the patient genotype, see example 4.
- viruses L210W, T215Y/F, K219Q) . Some viruses also contained mutations associated with resistance to ddl (V74I), ddC
- test virus (T69D) , or previously undescribed mutations (V75M, V75S, K219N) . Mutations for the test virus are listed below the test virus profile. Genotype described here is partial. For a complete description of the patient genotype, see example 5.
- V75I double site mutations were constructed by site directed mutagenesis. Phenotypic susceptibility to d4T and AZT of resistance test vectors containing these site directed mutations are shown. Left panel: The Q151M mutation reduced d4T susceptibility approximately three-fold. The V75I mutation did not alter d4T susceptibility. Right panel: The Q151M mutation reduced AZT susceptibility approximately five-fold. The V75I mutation increased AZT susceptibility approximately two-fold.
- FIG. 8 Site Directed Mutations: AZT Resistance-Associated Mutations. Resistance test vectors containing five (M41L, D67N, K70R, T215Y, K219Q) or six (M41L, D67N, K70R, L210W, T215Y, K219Q) AZT resistance associated mutations were constructed by site directed mutagenesis. Phenotypic susceptibility to d4T and AZT of resistance test vectors containing these site directed mutations are shown. Left panel: Resistance test vectors containing five or six AZT resistance-associated mutations were approximately twofold less susceptible to d4T than the control resistance test vector. Right panel: Resistance test vectors containing five or six AZT resistance-associated mutations were approximately 75-180-fold less susceptible to AZT than the control resistance test vector.
- Site Directed Mutations Single Mutations at RT Amino Acids 62, 69, and 75. Resistance test vectors containing single site (A62V, T69SSA, V75I, V75T) mutations were constructed by site directed mutagenesis. Phenotypic susceptibility to d4T and AZT of resistance test vectors containing these site directed mutations are shown. Left panel: The T69SSA and V75T mutations did not reduce d4T susceptibility appreciably (less than two-fold) . The A62V and V75I mutations had no affect on d4T susceptibility. Right panel: The V75I and V75T mutations increased AZT susceptibility slightly (approximately two-fold) . The
- Site Directed Mutations Multiple Mutations at RT Amino Acids 62, 69, and 75. Resistance test vectors containing double site (A62V+T69SSA) , (A62V+V75I) and triple site (A62V+T69SSA+V75I) mutations were constructed by site directed mutagenesis. Phenotypic susceptibility to d4T and AZT of resistance test vectors containing these site directed mutations are shown. Left panels: A combination of the A62V and T69SSA mutations did not reduce d4T susceptibility more than the T69SSA mutation alone. However, these two mutations reduced AZT susceptibility by approximately six-fold.
- a combination of the A62V and V75I mutations had no affect on d4T susceptibility.
- the A62V mutation did not alter the reduced level of AZT susceptibility caused by the V75I mutation.
- the V75I mutation completely suppressed the six-fold AZT resistance caused by the combination of A62V and T69SSA mutations .
- FIG. 11 Patient 285 clones: T69SSA Site Directed Revertant. Site directed mutagenesis was used to revert the T69SSA mutation in a molecular clone of a resistance test vector prepared from patient sample 285. The phenotypic susceptibility to d4T and AZT of the parental clone (T69SSA) and the revertant clone (SSA69T) are shown. Left panel: Reversion of the T69SSA mutation reduced d4T resistance by approximately three-fold. Right panel: Reversion of the T69SSA mutation reduced AZT resistance by approximately thirty-fold. Figure 12
- Patient 770 Clones +/- T69SSG+V75M.
- the resistance test vector pool derived from patient sample 770 was heterogeneous consisting of variants with or without the T69SSG and V75M mutations. The phenotypic susceptibility to d4T and AZT of resistance test vectors with or without these mutations are shown.
- Left panel Resistance test vector clones containing the T69SSG and V75M mutations were more than three-fold more resistant to d4T than clones without these mutations.
- Right panel Resistance test vector clones containing the T69SSG and V75M mutations were approximately thirty-fold more resistant to AZT than clones without these mutations.
- Site Directed Mutations Multiple Mutations at RT Amino Acids 41, 62, 69, 184 and 215.
- Phenotypic susceptibility to a panel of 6 NRTIs (AZT, ddC, DDI, 3TC, d4T and abacavir) of resistance test vectors containing these site directed mutations are shown.
- the M41L + T69SSA + T215Y significantly reduced susceptibility to all of the NRTIs tested (2-150 fold) .
- the addition of A62V resulted in a further reduction in susceptibility to AZT, d4T and ddl but had no effect on susceptibility to 3TC, ddC and abacavir.
- a resistance test vector with M41L + A62V + T69SSA + M184V + T215Y was more susceptible to AZT, d4T and ddl than the resistance test vector with the 4 mutations M41L + A62V + T69SSA + T215Y.
- the addition of M184V led to decreased susceptibility to 3TC but had no effect on susceptibility to ddC or abacavir.
- the present invention relates to methods of monitoring the clinical progression of HIV infection in patients receiving antiretroviral therapy, particularly nucleoside reverse transcriptase inhibitor antiretroviral therapy.
- the present invention provides for a method of assessing 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 RT having a mutation at one or more positions in the RT .
- the mutation (s) correlate positively with alterations in phenotypic susceptibility/resistance.
- the invention provides for a method of assessing the effectiveness of NRTI 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 RT having a mutation at codon 69.
- the invention established, using a phenotypic susceptibility assay, that mutations at codon 69 either alone or in combination with a mutation at codon 41 and 215 of HIV reverse transcriptase are correlated with a decrease in d4T susceptibility.
- the invention provides for a method of evaluating the effectiveness of NRTI 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 RT having one or more mutation (s) at codon (s) selected from the group consisting of 62, 75, 77, 116 and/or 151.
- This invention established, using a phenotypic susceptibility assay, that mutations at codons selected from the group consisting of 62, 75, 77, 116 and/or 151 either alone or in combination with one or more mutation (s) at codons selected from the group consisting of 41, 67, 210, 215, 219, 184, 69 and/or T215V of HIV reverse transcriptase are correlated with a decrease in d4T susceptibility (increased resistance) .
- the phenotypic susceptibility/resistance profile and genotypic profile of the HIV virus infecting the patient has been altered reflecting some change in the response to the antiretroviral agent.
- the HIV virus infecting the patient may be resistant to one or more but not another of the NRTIs as described herein. It therefore may be desirable after detecting the mutation, 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 NRTI antiretroviral agent and stopping d4T treatment; or (ii) increasing the dosage of d4T; or (iii) adding another antiretroviral agent to the patient's therapeutic regimen.
- the effectiveness of the modification in therapy may be 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.
- a method of assessing the effectiveness of NRTI therapy of a patient comprising (i) collecting a biological sample from an HIV-infected patient; (ii) amplifying the HIV-encoding RNA in the biological sample by converting the RNA to cDNA and amplifying HIV sequences using HIV primers that result in a PCR product that comprises the RT gene; (iii) performing PCR using primers that result in PCR products comprising wild type or mutant 69 and 41 and 215 codons; and (iv) determining, via the products of PCR, the presence or absence of a mutation at codon 69 or 41 or 215 or all three.
- a method of assessing the effectiveness of NRTI therapy of a patient comprising (i) collecting a plasma sample from an HIV- infected patient; (ii) amplifying the HIV-encoding RNA in the plasma sample by converting the RNA to cDNA and amplifying HIV sequences using HIV primers that result in a PCR product that comprises the RT gene; (iii) performing PCR using primers that result in PCR products comprising the wild type or mutations at codons selected from the group consisting of 62, 75, 77, 116 and 151 and/or one or more mutation (s) at codons selected from the group consisting of 41, 67, 210, 215, 219, 184, 69; and (iv) determining, via the products of PCR, the presence or absence of a mutation at codon 62, 75, 77, 116 and 151 and/or one or more mutation (s) at codons selected from the group consisting of
- a method of assessing the effectiveness of NRTI therapy of a patient comprising (i) collecting a plasma sample from an HIV-infected patient; (ii) amplifying the HIV-encoding RNA in the plasma sample by converting the RNA to cDNA and amplifying HIV sequences using HIV primers that result in a PCR product that comprises the RT gene; (iii) performing PCR using primers that result in PCR products comprising the wild type or mutations at codon 69 and 41, 210, 215, 184 or 74; and (iv) determining, via the products of PCR, the presence or absence of a mutation at codon 69 (T69SSA, T69SSG, T69SSS) and 41 (M41L) , 210 (L210W) , 215 (T215Y) , 184 (M184V) or 74 (L74V) .
- a method of assessing the effectiveness of NRTI therapy of a patient comprising (i) collecting a plasma sample from an HIV- infected patient; (ii) amplifying the HIV-encoding RNA in the plasma sample by converting the RNA to cDNA and amplifying HIV sequences using HIV primers that result in a PCR product that comprises the RT gene; (iii) performing PCR using primers that result in PCR products comprising the wild type or mutations at codon 41, 67, 70, 210, 215 and 219; and (iv) determining, via the products of PCR, the presence or absence of a mutation at codon 41 (m4lL) , 67 (D67N) , 70 (K70R) ; 210 (L210W) , 215 (T215Y/F) , and 219 (K219Q) .
- the presence of the mutation at codon 69 and 41 and 215 of HIV RT indicates that the effectiveness of the current or prospective NRTI therapy may require alteration, since as shown by this invention mutation at codon 69 reduces d4T susceptibility.
- mutation at codon 69 reduces d4T susceptibility.
- the presence of the mutation at codon (s) 62, 75, 77, 116 and/or 151 of the HIV RT indicates that the effectiveness of the current or prospective NRTI therapy may require alteration, since as shown by this invention mutation at codons 62, 75, 77, 116 and/or 151 reduces d4T susceptibility.
- the presence of the mutation/insertion at codon 69 (T69SSA, T69SSG, T69SSS) and 41 (M41L) , 210/L210W) , 215 (T215Y) , 184 (M184V) or 74 (L74V) of the HIV RT indicates that the effectiveness of the current or prospective NRTI therapy may require alteration, since as shown by this invention mutation/insertion at codon 69 either alone or in combination with mutation at codons 41, 210, 215, 184 or 74 reduces d4T susceptibility.
- 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 reverse transcriptase having a mutation at four or more codon (s) selected from the group consisting of 41, 67, 70, 210, 215 and 219 either alone or in combination with mutations at codon(s) 74 (V74I), 69 (T69D) , 75 (V75M, V75S) or 219 (K219N) .
- the reverse transcriptase has a mutation at codon V74I, T69D, V75M, V25S, K219N, or a combination thereof in addition to the four or more mutations at codons 41, 67, 70, 210, 215 and 219 of HIV RT.
- a method of assessing 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 reverse transcriptase having a mutation at one or more codons selected from the group consisting of 67, 75, 77, 116 and 151 and either alone or in combination with mutation at one or more codon (s) selected from the group consisting of (E6D, K20R, A33I, T39A, E44D, S68G, Y115F, I167V, E138A, G196A, I202V, T215V, D218E, and T240K) .
- This invention provides a method of assessing 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 reverse transcriptase having a mutation/insertion at codon 69 (T69SSA, T69SSG, T69SSS) either alone or in combination with mutations at one or more codons selected from the group consisting of V75M, A158S, K20R, V21I, K102M, V179I, V241L, 12831, E297R, E6D, Q174R, D177E, R284K, A288S, E291D.
- T69SSA nucleic acid encoding HIV reverse transcriptase having a mutation/insertion at codon 69
- This invention provides a method of assessing 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 reverse transcriptase having a mutation at four or more codons selected from the group consisting of 41, 67, 70, 210, 215 and 219 either alone or in combination with mutations at one or more codons selected from the group consisting of P1L, P9R, K20R, T39D, K43E, E44D, K64Y, V75M/S, G99R, L109V, V118I, K173E/T, I202T, R211H/T, D218E, K219N, H221Y, L228H, L283I, R284K, and A288T.
- This invention also provides the means and methods to u'se the resistance test vector comprising an HIV gene further comprising an NRTI mutation for drug screening. More particularly, the invention describes the resistance test vector comprising the HIV reverse transcriptase having mutations at codons 69 and 41 and 215 for drug screening. The invention also describes the resistance test vector comprising the HIV reverse transcriptase having mutations at codons selected from the group consisting of 62, 75, 77, 116 and 151 and/or 41, 67, 70, 210, 215 and 219. The invention also describes the resistance test vector comprising the HIV reverse transcriptase having mutations at four or more codons selected from the group consisting of 41, 67, 70, 210, 215 and 219.
- the invention further relates to novel vectors, host cells and compositions for isolation and identification of the nucleoside HIV-1 reverse transcriptase inhibitor resistance 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 reverse transcriptase of HIV-1 comprising contacting the compound with the polypeptide (s) comprising all or part of the HIV-1 reverse transcriptase, wherein codon 69 is changed to code for the insertion of amino acid residues SSS, SSG or SSA in place of threonine, wherein a positive binding indicates that the compound is capable of affecting the function of said reverse transcriptase.
- This invention provides a method for identifying a compound which is capable of affecting the function of HIV-1 reverse transcriptase comprising contacting the compound with a polypeptide (s) comprising all or part of the HIV-1 reverse ' transcriptase, wherein one or more codons selected from the group consisting of 62, 75, 77, 116 and 151 is changed to code for an amino acid residue (s) other than alanine, valine, phenylalanine, phenylalanine and glutamine respectively, wherein a positive binding indicates that the compound is capable of affecting the function of said reverse transcriptase.
- This invention also provides a method for identifying a compound which is capable of affecting the function of the reverse transcriptase of HIV-1 comprising contacting the compound with a polypeptide comprising a portion of the reverse transcriptase of HIV-1, wherein four or more codons selected from the group consisting of 41, 67, 70, 210, 215 and 219 are changed to code for an amino acid residues other than methionine, aspartic acid, lysine, leucine, threonine or lysine respectively, wherein a positive binding indicates that the compound is capable of affecting the function of said reverse transcriptase.
- 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 PCT International Application No. PCT/US97/01609, filed January 29, 1997 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.
- 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 may be underrepresented in one resistance test vector alone.
- Resistance test vector means one or more vectors which taken together contain DNA or RNA comprising a patient- derived segment and an indicator gene. Resistance test vectors are prepared as described in PCT International Application No. PCT/US97/01609, filed January 29, 1997 which is hereby incorporated by reference, 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.
- 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.
- “Indicator or indicator gene,” as described in PCT International Application No. PCT/US97/01609, filed January 29, 1997 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.
- the indicator or indicator gene may be functional or non-functional as described in PCT International Application No. PCT/US97/01609, filed January 29, 1997.
- 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 (i.e.
- viral drug resistance is a decrease in viral drug susceptibility for a given patient measured either by comparing the drug susceptibility to such ' a given standard or by making one or more sequential measurements in the same patient over time, as determined by increased inhibition of indicator gene expression (i.e. decreased indicator gene expression) .
- 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, and nelfinavir
- 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, nevirapine and delavirdine, 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 0.1 nM and about 100 ⁇ M and more specifically for each of the following drugs: AZT, from about 6 nM to about 400 ⁇ M; ddl, from about 15 nM to about 1,000 ⁇ M; 3TC, from about 9 nM to about 600E ⁇ M; d4T, from about 6 nM to about 400 ⁇ M; ddC, from about 15 nM to about 1,000 ⁇ M; nevirapine, from about 0.7 nM to about 50 ⁇ M; delavirdine, from about 0.07 nM to about 5 ⁇ M; saquinavir, from about 0.02 nM to about 1.5 ⁇ M; indinavir, from about 0.02 nM to about 1.5 ⁇ M; nelfinavir, from about 0.02 nM to about 1.5 ⁇ M; and ritonavir, from about 0.05
- a candidate NRTI antiretroviral compound is tested in the phenotypic drug susceptibility and resistance test using the resistance test vector comprising RT having mutations at codon 69 and 41 and 215.
- a candidate NRTI antiretroviral compound is tested in the phenotypic drug susceptibility and resistance test using the resistance test vector comprising RT having mutations at one or more codons selected from the group consisting of 62, 75, 77, 116 and/or 151.
- a candidate NRTI antiretroviral compound is tested in the phenotypic drug susceptibility and resistance test using the resistance test vector comprising RT having mutations at four or more codons selected from the group consisting M41L, D67N, K70R, L210W, T215Y/F and K219Q.
- a candidate NRTI antiretroviral compound is tested in the phenotypic drug susceptibility and resistance test using the resistance test vector comprising RT having mutations at codon 69 (either T69SSA, T69SSG, T69SSS) and M41L and T215Y and mutation at one or more codons selected from the group consisting of M184V/I, L74V, A62V, V75M.
- 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, delavirdine, nevirapine, saquinavir, ritonavir, indinavir, nelfinavir or a compound which is undergoing clinical trials such as abacavir, or a prenavir or efavirenz.
- 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.
- resistant mutants Following the identification of resistant mutants 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 NRTI antiretrovirals, alone or in combination. This will enable one of ordinary skill to identify effective NRTI antiretrovirals and design effective therapeutic regimens.
- 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 relegated 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.
- protein is removed by extraction with phenol/chloroform, and may be followed by ether extraction, and the nucleic acid recovered from aqueous fractions by precipitation with ethanol.
- 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 200C in 50 mM Tris (pH7.6) 50 mM NaCl, 6 mM MgC12, 6 mM DTT and 5-10 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-Cl pH 7.5, 10 mM MgCl2, 10 mM DTT, 33 mg/ml BSA, 10 mM- 50 mM NaCl, and either 40 ⁇ M ATP, 0.01-0.02 (Weiss) units T4 DNA ligase at OOC (for "sticky end” ligation) or ImM 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 1 ⁇ 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 protein 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.
- vectors containing 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) promoter system and hybrid promoters such as tac promoter.
- trp tryptophan
- other functional bacterial promoters are suitable.
- eukaryotic microbes such as yeast cultures may also be used.
- 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, or from heterologous mammalian promoters, e.g. ⁇ -actin promoter.
- viruses such as: polyoma, simian virus 40 (SV40) , adenovirus, retroviruses, hepatitis B virus and preferably cytomegalovirus, or from 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 which also contains the SV40 viral origin of replication.
- the immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindi I I restriction fragment.
- 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 (mdr) , the adenosine deaminase gene, and the glutamine 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) J. Molec. Appl . Genet.
- 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 transformation 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 and without antibiotics.
- 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.
- Phenotypic drug susceptibility and resistance tests are carried out using the means and methods described in PCT International Application No. PCT/US97/01609, filed January 29, 1997 which is hereby incorporated by reference .
- 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 + Pwo) , (Boehringer Mannheim. Indianapolis, IN) OR GeneAmp XL PCR kit (Tth + Vent) , (Roche Molecular Systems, Inc., Branchburg, NJ) ] .
- the primers, Apal primer (PDSApa) and Agel primer (PDSAge) used to amplify the "test" patient-derived segments contained sequences resulting in Apal and Agel recognition sites being introduced into the 5' and 3' termini of the PCR product, respectively as described in PCT International Application No. PCT/US97/01609, filed January 29, 1997.
- Resistance test vectors incorporating the "test" patient- derived segments were constructed as described in PCT International Application No. PCT/US97/01609, filed January 29, 1997 using an amplified DNA product of 1.5 kB prepared by RT-PCR ' using viral RNA as a template and oligonucleotides PDSApa (1) and PDSAge (2) as primers, followed by digestion with Apal and Agel or the isoschizimer PINAI .
- 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.
- 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 sequences that 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.
- the amount of luciferase activity produced in infected target host cells in the presence of drug was compared to the amount of luciferase produced in infected target host cells in the absence of drug.
- Drug resistance was measured as the amount of drug required to inhibit by 50% the luciferase activity detected in the absence of drug
- IC50 inhibitor concentration 50%, IC50
- Host cells were seeded in 10-cm-diameter dishes and were transfected several days after plating with resistance test vector plasmid DNA and the envelope expression vector. Transfections were performed using a calcium- phosphate 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.
- 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 loglO drug concentration. The 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 binding protein
- 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 2.
- 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 .
- 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 (drug susceptible) genetic background. Mutations may be incorporated alone and/or in combination with other known drug resistance 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 is 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.
- Resistance test vectors were constructed as described in example 1 from a patient samples designated as 96-136, 97- 240, 98-955 and 98-960. Patients 136 and 240 had been previously treated with regimens including d4T for various periods of time. The history of drug exposure for patients 955 and 960 is unknown. Isolation of viral RNA and RT/PCR was used to generate patient derived segments that comprised viral sequences coding for all of PR and aa 1 - 313 of RT . The patient derived segments were inserted into a indicator gene viral vector to generate resistance test vectors designated RTV-136, RTV-240. RTV-955 and RTV- 960.
- the RTVs were 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
- NNRTIs (AZT, 3TC, d4T, ddl, ddC, and abacavir), NNRTIs
- 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 decrease in susceptibility to d4T compared to a wild-type control RTV was observed in each of these RTV pools. The patient samples were examined further for genotypic changes that could be associated with the observed pattern of d4T susceptibility.
- RTV DNAs were 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.
- RTV-136 contained mutations at M41L, D67N, V75I, F116Y, Q151M, M184V, T200A, and T215Y. All mutations in RTV-136 were present as a mixture of wild-type and mutant amino acids at each position.
- RTV- 240 contained mutations at A62V, S68G, V75I, F77L, F116Y, E138A, Q151M, and M184V.
- RTV-955 contained mutations at E6D, K20R, V35I, A62V, D67N, T69D, V75I, F77L, K101E, K103N, Y115F, F116Y, Q151M, I167V, Y181C, M184V, G190A, I202V, R211K, F214L, T215V, and K219Q.
- the mutations at positions 101, 103, 181 and 190 were present as a mixture of wild-type and mutant amino acids at each position.
- RTV-960 contained mutations at A33I, T39A, M41L, E44D, D67N, T69D, K103N, Q151M, M184I, G190A, L210W, R211K, T215Y, D218E, T240K, and A288S.
- the mutations at A62V, V75I, F77L, F116Y, and Q151M have been previously described to result in broad spectrum cross resistance to NRTIs and are known as multi-drug resistance (MDR) mutations. All of these RTV DNAs contained one or several of these MDR mutations.
- RTV DNAs had mutations associated with AZT-resistance (M41L, D67N, L210W, T215Y, and K219Q) , ddC-resistance (T69D) , NNRTI- resistance (K101E, K103N, Y181C, and G190A) , 3TC- resistance (M184V) , or previously uncharacterized mutations (E6D, K20R, A33I, T39A, E44D, S68G, Y115F, I167V, E138A, G196A, I202V, T215V, D218E, and T240K) .
- the mutations at V35I, R211K, and F214L are known polymorphisms of the wild-type (drug-sensitive) variants of HIV.
- Site directed mutagenesis Resistance test vectors were constructed containing the Q151M mutation alone and in combination the V75I drug resistance mutations known to modulate the HIV susceptibility to NRTIs. 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). A resistance test vector containing the Q151M mutation (Q151M-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 151.
- the pattern of phenotypic susceptibility to the NRTI, d4T in the Q151M-RTV was altered as compared to wild type.
- the Q151M-RTV was less susceptible to d4T than the wild type control RTV.
- Significant changes in susceptibility to AZT, ddC and ddl were also observed in the Q151M-RTV.
- the Q151M mutation was also introduced into a RTV containing a mutation at V75I. The addition of the V75I mutation onto the Q151M background resulted in an increased susceptibility to AZT and a decreased susceptibility to d4T.
- Resistance test vectors were constructed as described in example 1 from patient samples designated 97-621, 97-285, 98-690, 98-770 and 98-771.
- Patient samples 285 and 690 are serial samples obtained from the same patient at 6 month intervals. Patients 285/690, 770 and 771 had been previously treated with regimens including d4T for various periods of time. The history of drug exposure for patient 621 is unknown.
- Patient VL#770 (JCW) was initially treated with AZT monotherapy. ddC was then added to the regimen, and the two drugs were given for a period of approximately 8 months. For a period of 15 months, the treatment was unknown and then patient VL#770 (JCW) was treated for approximately 2 1/2 years with a combination of AZT and 3TC.
- Patient VL# 285/690 (JWA) was treated with AZT monotherapy for approximately 1 year and 9 months before being switched to ddl monotherapy for a period of 8 months. He returned to AZT monotherapy for 1 year 3 months and then was treated with ddC for almost 2 years.
- the treatment was changed to a combination of AZT and 3TC for almost a year and then was changed to a combination of d4T/3TC/NFV for a period of approximately 9 months.
- Patient VL#771 (BMM) was initially treated with a combination of AZT, ddC and the PRI, RTV. 3TC was then added to this combination until treatment was switched to d4T and RTV. This treatment continued for approximately 6 months and was then changed to a combination of 3TC/d4T/ddI and RTV. There was a period of approximately 6 months in which no treatment was received. This was followed by treatment with AZT, 3TC and ddl. The combination was then switched to 3TC, d4T and IDV.
- Patient VL#1057 was treated with AZT monotherapy for approximately 3 years before being switched to ddl monotherapy for 1 year. There was a period of approximately 8 months in which no treatment was received. This was followed by treatment with AZT and 3TC for 1 year. The treatment with AZT continues but 3TC was replaced by d4T and the PRI, IDV was added. The d4T was then switched back to 3TC for a short period until it was replaced by the NNRTI, DEL. Isolation of viral RNA and RT/PCR was used to generate patient derived segments that comprised viral sequences coding for all of PR and aa 1 - 313 of RT.
- the patient derived segments were inserted into an indicator gene viral vector to generate resistance test vectors designated RTV-621, RTV-285.
- the RTVs were 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
- 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 decrease in susceptibility to d4T compared to a wild-type control RTV was observed in each of these RTV pools. The patient samples were examined further for genotypic changes that could be associated with the observed pattern of d4T susceptibility. Determination of genotype of patient RTV DNAs
- RTV DNAs were 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.
- RTV-621 contained mutations at M41L, A62V, T69SSS, V75M, M184V, L210W, and T215Y. Mutations in RTV-621 at positions 41, 62, and 75 were present as a mixture of wild-type and mutant amino acids at each position.
- RTV-285 contained mutations at M41L, A62V, T69SSA, L74V, A158S, I178M, M184V, T200A, L210W, and T215Y.
- RTV-690 contained mutations at K20R, M41L, A62V, T69SSA, L74V, A158S, I178M, Y181C, G190A, L210W, and T215Y.
- the mutations at positions 158 and 181 were present as a mixture of wild-type and mutant amino acids at each position.
- RTV-770 contained mutations at V21I, M41L, T69SSG, V75M, K102M, K103R, V179I, M184V, T215Y, V241L, L283I, and E297R.
- the mutations at positions 69 and 75 were present as a mixture of wild-type and mutant amino acids at each position.
- RTV-771 contained mutations at E6D, V35I, M41L, T69SSS, V75M, I135V, Q174R, D177E, M184V, T200I, L210W, R211K, T215Y, R284K, A288S, and E291D.
- the mutations at positions 174 and 200 were present as a mixture of wild-type and mutant amino acids at each position. All of these RTV DNAs had an unusual insertion of extra amino acids at or around position 69 in reverse transcriptase.
- the insertion is described here as T69SSX, where X indicates either a glycine (G) , a serine (S), or an alanine (A) .
- the mutation at A62V has been previously described as a member of the multi-drug resistance (MDR) mutations to contribute to broad spectrum cross resistance to NRTIs as described in example 3.
- MDR multi-drug resistance
- RTV DNAs had some subset of the mutations associated with AZT-resistance (M41L, D67N, L210W, T215Y, and K219Q) , ddC- resistance (L74V, L74I, T69D) , NNRTI-resistance (K103N, Y181C, and G190A) , ' 3TC-resistance (M184V) , or preiously uncharacterized mutations (V75M, A158S, K20R, V21I, K102M, V179I, V241L, 12831, E297R, E6D, Q174R, D177E, R284K, A288S, E291D) .
- T69SSX mutation in d4T resistance was further examined by the procedure commonly known as reverse mutagenesis.
- Site directed mutagenesis was used to specifically change the triplet "SSA" to a single threonine (T) at position 69.
- This revertant, 285- 1(SSA69T) contained all of the mutations present in the 285-1 clone except for the SSA insertion at position 69.
- the 285-1 (SSA69T) revertant showed a significant increase in susceptibility to both d4T (3 fold) and AZT (30 fold) .
- Further evidence for the role of the T69SSX insertion in NRTI resistance came from the examination of individual clones isolated from the RTV-770 pool. Two classes of clones were present in the RTV-770 pool: the first class contained mutations at V21I, M41L, K102M, K103R, V179I, M184V, T215Y, V241L, L283I, and E297R, the second class contained all of the mutations present in the first class and in addition had mutations at T69SSG and V75M. The second class of mutations, those with mutations at 69 and 75, showed significant increases in susceptbility to both d4T (4 fold) and AZT (30 fold) .
- Site directed mutagenesis Resistance test vectors were constructed containing the T69SSA mutation alone and in combination with other drug resistance mutations known or suspected to modulate the HIV susceptibility to NRTIs. 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).
- a resistance test vector containing the T69SSA mutation (T69SSA-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 (T) at position 69.
- the T69SSA-RTV showed a 2-fold decrease in susceptibility to d4T compared to a wild type RTV. A small but significant change in susceptibility to AZT was also observed in the T69SSA-RTV.
- the T69SSA mutation was also introduced into a RTV containing a mutation at V75I.
- the addition of the V75I mutation onto the T69SSA background resulted in an increased susceptibility to AZT and a decreased susceptibility to d4T.
- the V75I mutation alone had no effect on d4t-susceptibility and resulted in an increase in the susceptibility to AZT.
- the T69SSA mutation was also introduced into a RTV containing a mutation at A62V.
- the A62V mutation alone had no effect on the susceptibility to any of the RT inhibitors tested.
- the addition of the A62V mutation onto the T69SSA background had no effect on the susceptibility to d4T but resulted in a significant decrease in susceptibility to AZT (6 fold) .
- a RTV was constructed containing mutations at A62V, T69SSA, and V75I.
- the triple mutant showed only a very small decerase in susceptibility to AZT (2 fold) (the same as T69SSA alone) and a small (less than 2 fold) increase in susceptibility to AZT.
- RTVs were constructed that contained T69SSA insertion in conjunction with the AZT-resistance mutations M41L, A62V, T215Y and the 3tc-resistance mutation M184V in various combinations.
- the RTV containing mutaions at M41L, T69SSA and T215Y showed a significant decrease in susceptibility to both d4T (5 fold) and AZT (160 fold) .
- the addition of an A62V mutation onto this background further decreased the susceptibility to both d4T (10 fold) and AZT (>1000 fold) .
- the presence of the M184V mutation had no effect on the susceptibility to d4T but causes an increase in the susceptibility to AZT.
- a resistance test vector was constructed containing mutations at M41L, T69SSA, T215Y and L210W.
- the introduction of the L210W mutation into an RTV containing the three mutations _ (M41L, T69SSA, and T215Y) resulted in a substantial decrease in susceptibility to AZT (greater than 1000-fold) compared to the 140-fold decrease in susceptibility observed for AZT in M41L-T69SSA-T215Y-RTV.
- the L210W mutation had little effect on the susceptibility to the other NRTIs when compared to M41L-T69SSA-T215Y-RTV.
- M41L-T69SSA-T215Y-L210W-RTV changed only slightly when compared to M41L-T69SSA-T215Y-RTV.
- the L210W mutation had no additional effect on susceptibility to the NNRTIs, DEL (0.13-fold) and NEV (0.42-fold), compared to M41L-T69SSA-T215Y-RTV, which also displayed a slight increase in susceptibility to DEL and NEV.
- a RTV was constructed which contained four mutations: M41L, A62V, T69SSA, and T215Y.
- This vector, M41L-A62V- T69SSA-T215Y-RTV displayed a substantial decrease in susceptibility to AZT (greater than 1000-fold) , wild-type susceptibility to ddC (2.1-fold), a slight decrease in susceptibility to ddl (3-fold), a slight decrease in susceptibility to 3TC (8-fold), a slight decrease in susceptibility to d4T (8-fold), and a slight decrease in susceptibility to abacavir (10-fold) .
- the vector displayed an increase in susceptibility to the NNRTIs, DEL (0.2-fold) and NEV (0.8-fold).
- the L210W mutation was also introduced into the vector containing four other mutations: M41L, A62V, T69SSA, and T215Y.
- the vector displayed an increase in susceptibility to the NNRTIs, DEL (0.2-fold) and NEV (0.6-fold).
- the susceptibilities observed for M41L-A62V-T69SSA-T215Y-L210W-RTV were similar to those observed for the vector containing the M41L, A62V, T69SSA, and T215Y mutations but lacking the L210W mutation.
- the T215Y mutation was introduced into a vector containing the A62V and T69SSA mutations.
- the introduction of the T215Y mutation resulted in a substantial decrease in susceptibility to AZT (1000-fold) compared to A62V-T69SSA- RTV, which displayed only a slight decrease in susceptibility to AZT (7-fold) .
- the susceptibility to the other inhibitors was similar to the susceptibilities observed for the vector without the T215Y mutation.
- the A62V-T69SSA-T215Y-RTV displayed wild-type susceptibility to ddC (1.3-fold), a slight decrease in susceptibility to ddl (2.5-fold), a moderate decrease in susceptibility to 3TC (15-fold) , a slight decrease in susceptibility to d4T (7-fold) , and a slight decrease in susceptibility to abacavir (10-fold).
- the vector displayed increases in susceptibility to the both NNRTIs, DEL (0.3-fold) and NEV (0.6-fold) .
- the L74V mutation was also introduced into a vector containing the A62V and T69SSA mutations.
- the susceptibility to the inhibitors tested was similar to the susceptibilities observed for the vector without the L74V mutation and similar to the vector containing the T215Y mutation along with the A62V and T69SSA mutations.
- the greatest change was a shift of the susceptibility back to wild-type observed for AZT in A62V-T69SSA-L74V-RTV.
- the A62V-T69SSA-L74V-RTV displayed wild-type susceptibility to ddC (1.8-fold), wild-type susceptibility to ddl (1.9- fold), a slight decrease in susceptibility to 3TC (2.5- fold) , wild-type susceptibility to d4T (1.5-fold), and a slight decrease in susceptibility to abacavir (3-fold) .
- the vector displayed slight increases in susceptibility to the both NNRTIs, DEL (0.4-fold) and NEV (0.3-fold).
- a RTV was constructed containing mutations at M41L, T69SSA, T215Y L210W and V75M.
- M41L-T69SSA-T215Y-L210W-V75M- RTV displayed a substantial decrease in susceptibility to AZT (greater than 1000-fold) , a slight decrease in susceptibility to ddC (2.9-fold), a slight decrease in susceptibility to ddl (3.5-fold), a moderate decrease in susceptibility to 3TC (17-fold), a moderate decrease in susceptibility to d4T (11-fold), and a moderate decrease in susceptibility to abacavir (19-fold).
- the vector displayed an increase in susceptibility to the NNRTIs, DEL (0.25-fold) and NEV (0.5-fold).
- Phenotypic analysis of Resitance Test Vectors from Patients 98-757, 98-844, 98-937, 98-964 and 98-966 Resistance test vectors were constructed as described in example 1 from a patient samples designated 98-757, 98- 844, 98-937, 98-964 and 98-966. All of these patients had been previously treated with regimens including d4T for various periods of time. Isolation of viral RNA and RT/PCR was used to generate patient derived segments that comprised viral sequences coding for all of PR and aa 1 - 313 of RT . The patient derived segments were inserted into a indicator gene viral vector to generate resistance test vectors designated RTV-757, RTV-844.
- RTV-937, RTV-964 and RTV-966 were 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 and nevirapine), and PRIs (indinavir, nelfinavir, ritonavir, and saquinavir) .
- 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 decrease in susceptibility to d4T compared to a wild-type control RTV was observed in each of these RTV pools. The patient samples were examined further for genotypic changes that could be associated with the observed pattern of d4T susceptibility.
- RTV DNAs were 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.
- RTV-757 contained mutations at V35I, D67N, T69D, K70R, V106A, V108I, L109V, Y181C, V189L, T200A, I202T, R211K, T215F, D218E, K219Q, H221Y, L228H, L283I, and R284K.
- RTV-844 contained mutations at M41L, D67N, I135V, L210W, T215Y, and K219N.
- RTV-937 contained mutations at P1L, P9R, K20R, V35T, K64Y, D67N, K70R, V75M, G99R, I135V, K173E, Y188L, R211H, T215F, D218E, and K219Q.
- RTV-964 contained mutations at M41L, K43E, D67N, K70R, L74I, V75S, Y181I, R211T, T215Y, D218E, and K219Q.
- RTV-966 contained mutations at K20R, T39D, M41L, E44D, D67N, L74V, A98S, V118I, I135T, K166R, K173T, M184V, G196E, L210W, R211K, T215Y, D218E, K219R, L228H, V245E, K277R, T286A, A288T, V293I. All of these RTV DNAs contained one or several mutations previously associated with AZT-resistance (M41L, D67N, L210W, T215F/Y, and
- V106A, V108I, Y181C/I, and Y188L 3TC-resistance (M184V) , or previously uncharacterized mutations (P1L,
- V35I/T, A98S, I135V/T, K166R, G196E, T200A, R211K, F214L, V245E, K277R, T286A, and V293I are known polymorphisms of the wild-type (drug-sensitive) variants of HIV.
- Site directed mutagenesis Resistance test vectors containing five (M41L, D67N, K70R, T215Y, K219Q) or six (M41L, D67N, K70R, L210W, T215Y, K219Q) AZT-resistance associated mutations were constructed by site directed mutagenesis. Phenotypic susceptibility to NRTIs were determined. Significant decreases in susceptiblity to AZT (75 - 180 fold) and d4T (2 fold) were observed in these RTVs.
- changes in the amino acid at position 69 of the reverse transcriptase 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 reverse transcriptase having a mutation at codon 69.
- a reduction in d4T susceptibility and decreased AZT susceptibility is correlated with the presence of a mutation at codon 69 (T69SSX) either alone or on a background of other NRTI-resistance mutations (for example M41L, A62V, D67N, K70R, L74V, V75I/M, T215Y/F, K219Q) .
- 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
- 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 69 of the HIV-1 reverse transcriptase is mutated, can be performed using various methods, such as direct characterization of the viral nucleic acid encoding reverse transcriptase or direct characterization of the reverse transcriptase protein itself. Defining the amino acid at position 69 of reverse transcriptase can be performed by direct characterization of the reverse transcriptase 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 69 of the HIV-1 reverse transcriptase protein can be defined by characterizing amplified copies of HIV-1 nucleic acid encoding the reverse transcriptase protein.
- Amplification of the HIV-1 nucleic acid can be performed using a variety of methodologies including reverse transcription- poly erase chain reaction (RT-PCR) , NASBA, SDA, RCR, or 3SR as would be known to the ordinarily skilled artisan.
- Evaluating whether the nucleic acid encoding HIV reverse transcriptase has a mutation at codon 69 can be performed 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
- primer extension-chain termination Sanger, ABI/PE and Visible Genetics
- chain cleavage Maxam and Gilbert
- sequencing methods such as matrix assisted laser desorption- ionization time of flight (MALDI-TOF) or mass spectrometry
- nucleic acid sequence encoding amino acid position 69 can be evaluated using a variety of probe hybridization methodologies, such as genechip hybridization sequencing
- evaluation of whether amino acid position 69 of HIV-1 reverse transcriptase was wild type or mutant was carried out using a phenotypic susceptibility assay 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 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 patient samples having a T69SSX mutation/insertion is shown in Figure 5.
- the nucleic acid (DNA) sequence of the patient derived HIV-1 protease and reverse transcriptase regions from patient samples 621, 690, 770, 771 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 susceptibility profiles of patient samples and site directed mutants showed a significant reduction m d4T susceptibility (increased resistance) AZT susceptibility correlated with a mutation m the nucleic acid sequence encoding the ammo acids serine, serine, serine (SSS), serine, serine, alanine (SSA) or serine, serine, glycine (SSG) at position 69 of HIV-1 reverse transcriptase and the presence of mutations at some subset of the positions described above.
- positions include those previously associated with NRTI-resistance (41, 67, 70, 74, 75, 184, 210, 215, and 219) or the positions observed to be mutated m these patients which have been previously uncharaqterized (1, 6, 9, 20, 21, 33, 39, 43, 44, 64, 68, 99, 109, 115, 118, 138, 158, 167, 173, 174, 177, 179, 196, 202, 211, 218, 221, 228, 240, 241, 283, 284, 288, 291, 297)
- Phenotypic susceptibility profiles of patient samples with insertions at 69 showed decreased susceptibility to AZT, 3TC, ddC, ddl, d4T, and abacavir.
- Phenotypic and genotypic correlation of mutations at amino acids 62, 75, 77, 116 and 151 of HIV-1 Reverse Transcriptase Phenotypic and genotypic correlation of mutations at amino acids 62, 75, 77, 116 and 151 of HIV-1 Reverse Transcriptase .
- Phenotypic susceptibility profiles of patient samples and site directed mutants showed decreases susceptibility to NRTIs (AZT, ddC, ddl, 3TC, d4T and abacavir) when the positions 62, 75, 77, 116 and 151 or some subset of those positions contained ammo acids 62V, 751, 77L, 116F, or 151M HIV-1 reverse transcriptase.
- the presence of additional mutations at positions 41, 67, 69, 184, 210, 215 and 219 could further modify (increase or decrease) susceptibility to the NRTIs.
- Phenotypic susceptibility profiles of patient samples and site directed mutants showed significant decreases in susceptibility to d4T when mutations were present at positions previously correlated with loss of susceptibility to AZT (41, 67, 70, 210, 215, and 219) and also at some subset of positions previously correlated with loss of susceptibility to other NRTIs (74, 75, 184) or with previously uncharacterized mutations observed in the patient viruses described above (1, 6, 9, 20, 21, 33, 39, 43, 44, 64, 68, 99, 109, 115, 118, 138, 158, 167, 173, 174, 177, 179, 196, 202, 211, 218, 221, 228, 240, 241, 283, 284, 288, 291, 297) .
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Cited By (18)
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WO2001079540A2 (en) | 2000-04-18 | 2001-10-25 | Virco Bvba | Methods for measuring 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 |
US6958211B2 (en) | 2001-08-08 | 2005-10-25 | Tibotech Bvba | Methods of assessing HIV integrase inhibitor therapy |
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 |
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 |
US7494768B1 (en) | 1999-05-28 | 2009-02-24 | Tibotec-Virco Virology Bvba | Mutational profiles in HIV-1 protease and reverse transcriptase 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 |
US7579143B2 (en) | 1998-05-26 | 2009-08-25 | Monogram Biosciences, Inc. | Method of assessing the effectiveness of a non-nucleoside reverse transcriptase inhibitor (NNRTI) on a human immunodeficiency virus type 1 (HIV-1)-infected patient |
AU2007203337B2 (en) * | 2000-04-18 | 2009-12-17 | Virco Bvba | Methods for measuring 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 |
US8071284B2 (en) | 2005-06-06 | 2011-12-06 | Monogram Biosciences, Inc. | Methods and compositions for determining altered susceptibility of HIV-1 to anti-HIV drugs |
US8076062B2 (en) | 2002-07-01 | 2011-12-13 | Tibotec Pharmaceuticals Ltd. | Mutational profiles in HIV-1 protease correlated with phenotypic drug resistance |
US8178291B2 (en) | 2005-02-18 | 2012-05-15 | Monogram Biosciences, Inc. | Methods and compositions for determining hypersusceptibility of HIV-1 to non-nucleoside reverse transcriptase inhibitors |
EP2458019A2 (en) | 2005-05-27 | 2012-05-30 | Monogram BioSciences, Inc. | Methods and compositions for determining resistance of HIV-1 to protease inhibitors |
US8574831B2 (en) | 2000-10-20 | 2013-11-05 | Virco N.V. | Mutational profiles in HIV-1 reverse transcriptase correlated with phenotypic drug resistance |
US8592161B2 (en) | 2002-07-01 | 2013-11-26 | Janssen R&D Ireland | Mutational profiles in HIV-1 protease correlated with phenotypic drug resistance |
US10480037B2 (en) | 2011-09-23 | 2019-11-19 | Laboratory Corporation Of America Holdings | Methods and systems for predicting HIV-1 coreceptor tropism |
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CN103965302B (en) * | 2013-01-29 | 2019-05-28 | 军事科学院军事医学研究院微生物流行病研究所 | A kind of recombination super antigen SEB mutant, preparation method and application |
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AU719691B2 (en) * | 1996-01-26 | 2000-05-18 | Innogenetics N.V. | Method for detection of drug-induced mutations in the reverse transcriptase gene |
PL328068A1 (en) * | 1996-01-29 | 1999-01-04 | Virologic | Compositions for and methods of determining susceptibility to and immunity against an antiviral drug and selecting appropriate antiviral drug |
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1999
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- 1999-06-24 WO PCT/US1999/014486 patent/WO1999067427A1/en active IP Right Grant
- 1999-06-24 MX MXPA00012843A patent/MXPA00012843A/en unknown
- 1999-06-24 EP EP99933581A patent/EP1015642A4/en not_active Withdrawn
- 1999-06-24 JP JP2000556067A patent/JP2003503004A/en active Pending
- 1999-06-24 AU AU49611/99A patent/AU769927B2/en not_active Ceased
- 1999-06-24 CN CNB998134791A patent/CN1154747C/en not_active Expired - Fee Related
- 1999-06-24 BR BR9912209-0A patent/BR9912209A/en not_active IP Right Cessation
- 1999-06-24 NZ NZ508981A patent/NZ508981A/en unknown
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US7494768B1 (en) | 1999-05-28 | 2009-02-24 | Tibotec-Virco Virology Bvba | Mutational profiles in HIV-1 protease and reverse transcriptase correlated with phenotypic drug resistance |
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WO2001079540A3 (en) * | 2000-04-18 | 2003-06-05 | Virco Bvba | Methods for measuring drug resistance |
JP2004500836A (en) * | 2000-04-18 | 2004-01-15 | ビルコ・ビーブイビーエイ | How to measure drug resistance |
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US8076062B2 (en) | 2002-07-01 | 2011-12-13 | Tibotec Pharmaceuticals Ltd. | 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 |
US8592161B2 (en) | 2002-07-01 | 2013-11-26 | Janssen R&D Ireland | Mutational profiles in HIV-1 protease correlated with phenotypic drug resistance |
US8178291B2 (en) | 2005-02-18 | 2012-05-15 | Monogram Biosciences, Inc. | Methods and compositions for determining hypersusceptibility of HIV-1 to non-nucleoside reverse transcriptase inhibitors |
US10202658B2 (en) | 2005-02-18 | 2019-02-12 | Monogram Biosciences, Inc. | Methods for determining hypersusceptibility of HIV-1 to non-nucleoside reverse transcriptase inhibitors |
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US8071284B2 (en) | 2005-06-06 | 2011-12-06 | Monogram Biosciences, Inc. | Methods and compositions for determining altered susceptibility of HIV-1 to anti-HIV drugs |
US8637252B2 (en) | 2005-06-06 | 2014-01-28 | Monogram Biosciences, Inc. | Methods and compositions for determining altered susceptibility of HIV-1 to anti-HIV drugs |
US10480037B2 (en) | 2011-09-23 | 2019-11-19 | Laboratory Corporation Of America Holdings | Methods and systems for predicting HIV-1 coreceptor tropism |
Also Published As
Publication number | Publication date |
---|---|
EP1015642A4 (en) | 2005-03-23 |
AU769927B2 (en) | 2004-02-12 |
MXPA00012843A (en) | 2002-04-24 |
EP1015642A1 (en) | 2000-07-05 |
JP2003503004A (en) | 2003-01-28 |
AU4961199A (en) | 2000-01-10 |
BR9912209A (en) | 2002-11-05 |
CA2341679A1 (en) | 1999-12-29 |
CN1154747C (en) | 2004-06-23 |
WO1999067427A9 (en) | 2000-03-30 |
CN1332804A (en) | 2002-01-23 |
NZ508981A (en) | 2004-02-27 |
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