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WO2000066774A2 - Analyse amelioree et ses reactifs - Google Patents

Analyse amelioree et ses reactifs Download PDF

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Publication number
WO2000066774A2
WO2000066774A2 PCT/GB2000/001639 GB0001639W WO0066774A2 WO 2000066774 A2 WO2000066774 A2 WO 2000066774A2 GB 0001639 W GB0001639 W GB 0001639W WO 0066774 A2 WO0066774 A2 WO 0066774A2
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virus
viral
deletion
dna vector
hiv
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PCT/GB2000/001639
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English (en)
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WO2000066774A3 (fr
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Edward Duncan Blair
Laurence Henry Robinson
Barbara Wendy Snowden
Sylvia Margaret Tisdale
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Glaxo Group Limited
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Priority to AU45867/00A priority Critical patent/AU4586700A/en
Priority to EP00927464A priority patent/EP1226270A2/fr
Priority to JP2000615396A priority patent/JP2002542804A/ja
Publication of WO2000066774A2 publication Critical patent/WO2000066774A2/fr
Publication of WO2000066774A3 publication Critical patent/WO2000066774A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • C12Q1/701—Specific hybridization probes
    • C12Q1/702—Specific hybridization probes for retroviruses
    • C12Q1/703—Viruses associated with AIDS
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to methods for generating recombinant viruses from samples such as uncharacterised virus samples or clinical specimens, to the use of the viruses so generated in assays, predominantly for the purpose of detecting altered viral susceptibility to anti-viral drugs, and to reagents, more particularly deoxyribonucleic acid (DNA) vector constructs, for use in such methods and assays.
  • the assays are adapted to detect resistant virus more accurately and sensitively than known assays by taking into account compensatory mutations arising in nucleotide sequences other than those encoding the anti-viral drug target.
  • HIV-1 human immunodeficiency virus type 1
  • anti-retroviral drugs presents a major challenge in the chemotherapeutic prevention of progression to Acquired Immunodeficiency Syndrome (AIDS).
  • AIDS Acquired Immunodeficiency Syndrome
  • RT viral reverse transcriptase
  • the current goal of anti-HIV therapy is maximally to suppress replication of the virus so as to delay the appearance of drug-resistant variants and maintain healthy levels of CD4 + immune cells for as long as possible (Vandamme et al., 1998).
  • the more recently introduced therapies of HIV infection usually involve combinations of three more anti-retroviral drugs.
  • RTIs reverse transcriptase inhibitors
  • Pro or PR inhibitors of the viral protease enzyme
  • protease inhibitors or Pis protease inhibitors or Pis
  • RT and Pro HIV-1 viruses with resistance mutations in either or both drug of the targets RT and Pro
  • These mutations alter the structure and/or chemical affinities of the target enzymes such that their ability to interact with the drugs is altered or reduced and the drugs show diminished activity against the mutated virus.
  • Drug resistance mutations can potentially occur in the drug target molecules of any other viruses.
  • antiviral drugs may act on the viral protease or, if the virus is an RNA virus, at the reverse transcriptase, other targets may also be employed.
  • any viral nucleic acid or protein critical to viral reproduction or infectivity may be a potential drug target, for example several anti-herpes nucleoside analogue drugs e.g. aciclovir act against HSV 1 or 2, Varicella Zoster and other herpes family viruses through phosphorylation of the drug molecule by the viral thymidine kinase and further processing by host cellular enzymes, including incorporation into viral DNA by host DNA polymerase activity. Mutations associated with reduced susceptibility to nucleoside analogues have been demonstrated in the thymidine kinases or DNA polymerases of herpes viruses (Kimberlin and Whitley, 1996; Balfour, 1999). Other viral drug targets include the DNA maturation factors and DNA polymerases of certain DNA viruses.
  • CSs cleavage sites
  • the p7 protein is cleaved from the Gag and Gag-Pol precursor polyproteins at the p7/p1 cleavage site (CS); viral proteins p1 and p6 are cleaved from the Gag polyprotein at the p1/p6 CS; Pro, RT and integrase (INT) are also produced from cleavage of Gag-Pol.
  • Mutations at cleavage sites are thought to compensate for impaired polypeptide cleavage activity of the mutant Pro which would otherwise lead to loss of viral fitness and are hereafter referred to as compensatory mutations.
  • Compensatory mutations have been documented at the p7/p1 and the p1/p6 CSs (Doyon et al., 1996; Maschera et al., 1996a, b; Zhang et al., 1997; Carrillo et al., 1998; Mammano et al., 1998; Zennou et al., 1998).
  • mutations associated with reduced susceptibility to currently marketed anti-retroviral agents occur in at least three distinct regions of the HIV-1 genome: RT, Pro and the CSs.
  • PBMC peripheral blood mononuclear cells
  • Co-culture of PBMC is not ideal for large scale regular application because it involves the isolation of fresh PBMC and the long culture times have been shown to select for minority or less drug-resistant variants (Kusumi et al., 1992; Mayers et al., 1998).
  • the recombinant virus assay (RVA) enables the rapid and reproducible determination of phenotypic susceptibility of HIV-1 from plasma (Kellam and Larder, 1994; Maschera ef al., 1995; Hertogs et al., 1998) and has thus been instrumental in directing the choice of drugs used in HIV-therapy.
  • HIV RT or Pro sequences are amplified from plasma by reverse transcription-polymerase chain reaction (RT-PCR) and co- electroporated into CD4 + cultured cells (MT4) with a molecular clone of an RT or Pro-deleted "provirus" of the HIV-1 standard laboratory strain wild type HXB2, termed the 'vector'.
  • RT-PCR reverse transcription-polymerase chain reaction
  • MT4 + cultured cells a molecular clone of an RT or Pro-deleted "provirus” of the HIV-1 standard laboratory strain wild type HXB2, termed the 'vector'.
  • Provirus is the term used to describe the DNA copy of an HIV virus genome which integrates into the host cell's DNA.
  • RT or PR sequences insert into the corresponding deletion site of the wild type vector by homologous recombination, and the resulting recombinant vector DNA inserts into the cellular DNA to yield full length infectious proviruses.
  • Gene expression from the proviruses yields a population of viruses with a drug susceptibility phenotype representative of the subject's plasma viruses.
  • the resulting virus stocks can be used for drug sensitivity tests against PR or RT inhibitors, depending on which part of the genetic information in the recombinant virus is derived from the clinical isolate.
  • the RVA has several advantages over the co-culture of PBMC in addition to its rapidity and reproducibility.
  • the production of virus in a common backbone enables a more accurate comparison of the drug sensitivities and growth characteristics of the viruses produced, and the shorter culture times minimise the outgrowth of minor variants.
  • RVA-type assays can be directed to the detection of drug-resistant mutants of viruses other than HIV, by the recombination of sequences corresponding to the anti-viral drug target, derived by PCR or RT-PCR from a patient tissue sample, with a DNA vector including a wild type (or other standard laboratory strain) viral genome carrying a deletion corresponding to the sequence encoding the drug target.
  • the present invention provides an assay for the detection of virus resistant to an anti-viral drug by recombination of a DNA vector with a nucleotide sequence derived from a sample of virus suspected of including drug-resistant virus to produce viable recombinant virus having the resistance profile of the virus sample, wherein the DNA vector comprises a wild- type laboratory virus strain genome carrying a deletion of at least a portion of the sequence encoding the antiviral drug target and a further deletion of sequences comprising a potential site of compensatory mutation.
  • the nucleotide sequence derived from the sample of virus which may be obtained by PCR, RT-PCR, or related methods, comprises a region of the viral genome substantially corresponding to the sequences deleted from the DNA vector.
  • the invention provides an assay for the detection of virus resistant to an anti-viral drug comprising the step of generating recombinant virus having a drug resistance profile of the virus in a viral sample by recombination of a DNA vector with a nucleotide sequence derived from the sample, wherein the DNA vector comprises a wild-type laboratory virus strain genome carrying a deletion of at least a portion of the sequence encoding the antiviral drug target and a further deletion of sequences comprising a potential site of compensatory mutation.
  • the assay may further comprise the steps of infecting cells with recombinant virus so produced, incubating infected cells with an antiviral drug and detecting the viability of virus or of infected cells after incubation to determine the sensitivity of the recombinant virus to the drug.
  • the drug target is a viral protease, polymerase or reverse transcriptase enzyme.
  • the virus is a herpes family virus or an
  • the DNA vector comprises the sequence of an HIV provirus carrying a deletion in a gene encoding a drug target and in a site of compensatory mutation.
  • the DNA vector carries a deletion in the sequence encoding the viral protease and a further deletion of a sequence encoding one or more protease cleavage sites.
  • the DNA vector carries a deletion of one or both of the HIV-1 p7/p1 and p1/p6 protease cleavage sites in addition to a deletion of the sequence encoding the viral protease, or a fragment thereof.
  • the assay employs a DNA vector carrying a deletion of at least a fragment of the sequence encoding the viral reverse transcriptase.
  • sequences encoding the viral reverse transcriptase and the viral protease, or fragments thereof are deleted in addition to a deletion of one or more protease cleavage sites.
  • the DNA vector may carry a deletion of one or both of the HIV-1 p7/p1 and p1/p6 protease cleavage sites, of the entire sequence encoding the viral protease, of the entire sequence encoding the viral reverse transcriptase and, optionally, of the sequence encoding the viral polymerase, or a fragment thereof.
  • the present invention provides DNA vectors comprising a wild-type laboratory virus strain genome carrying a deletion of at least a portion of the sequence encoding an antiviral drug target and a further deletion of sequences comprising a site of compensatory mutation.
  • the drug target is a viral protease, polymerase or reverse transcriptase enzyme.
  • the virus is a herpes family virus or an HIV virus, preferably an HIV virus, more preferably HIV-1 , in which case the DNA vector comprises the sequence of an HIV-1 provirus carrying a deletion in a sequence encoding a drug target and in a site of compensatory mutation.
  • the vector carries a deletion in the sequence encoding the viral protease and a further deletion of a sequence encoding one or more protease cleavage sites.
  • the vector carries a deletion of one or both of the HIV-1 p7/p1 and p1/p6 protease cleavage sites in addition to a deletion of the sequence encoding the viral protease, or a fragment thereof.
  • the DNA vector carries a deletion of at least a fragment of the sequence encoding the viral reverse transcriptase.
  • sequences encoding the viral reverse transcriptase and the viral protease, or fragments thereof are deleted in addition to a deletion of one or more protease cleavage sites.
  • the DNA vector may carry a deletion of one or both of the HIV-1 p7/p1 and p1/p6 protease cleavage sites, of the entire sequence encoding the viral protease, of the entire sequence encoding the viral reverse transcriptase and, optionally, of the sequence encoding the viral polymerase, or a fragment thereof.
  • the present provides DNA vectors such as set out above for use in an assay according to the first aspect of the present invention.
  • compensatory mutations may occur in viral proteins associated with the RT enzyme in its functional role (accessory proteins), or in the binding, activation or initiation sites in the RNA genome from which the RT enzyme commences reverse transcription of the genome in the first step of viral replication.
  • the viral polymerase enzyme is the drug target, in which circumstances resistance mutations may occur in the viral sequences encoding the polymerase and compensatory mutations might be envisaged in accessory proteins or in polymerase binding, activation, initiation etc. sequences of the viral genome.
  • viruses could overcome drug induced mutations which are deleterious to viral growth include the modulation of expression of those drug targets by compensatory mutations in regulatory nucleic acid sequences or by mutations affecting viral factors which control levels, timings or patterns of expression.
  • Compensatory mutations in regulatory sequences may or may not cause amino acid changes in any proteins they encode.
  • the skilled man will be able to modify the teachings of the present invention to create DNA vectors for use in RVA assays in which the sites of such compensatory mutations are deleted, in addition to deletion of RT, Pro, polymerase or other drug target sequence (or a region thereof). Such vectors are included within the present invention.
  • sample (RT-)PCR products derived from infected subjects may be co-transfected with a vector derived from any previously characterised strain of HIV-1 to produce viable recombinant virus, for example strains other than the standard HXB2 laboratory strain may be used.
  • strains other than the standard HXB2 laboratory strain
  • the strain chosen to provide the vector sequence should preferably be well characterised in order that variations in growth and sensitivity to antivirals in the recombinant virus produced can be assigned to the vector strain or to the sequences derived from the viral (e.g. patient) sample.
  • a wild-type strain is generally suitable as it contains no pre-existing resistance mutations.
  • the vector strain also needs to be chosen with regard to the cell culture conditions to be used in the generation of recombinant virus and the subsequent drug resistance. The skilled man will be able to select a vector strain which displays sufficiently strong replication characteristics in the cell culture of interest.
  • reference to a "laboratory strain”, “laboratory virus strain”, “wild type strain” or to a "wild-type laboratory virus strain” should be understood to mean any previously characterised viral strain.
  • the present invention provides a kit for the performance of an assay according to the first aspect of the invention, the kit comprising a DNA vector construct according to the second aspect of the invention. Also provided is the use of a DNA vector construct according to the present invention in an assay for the detection of virus resistant to an anti-viral drug.
  • FIG. 1. illustrates diagrammatically the construction of three CS-deleted plasmids for the RVA
  • (A) shows the plBI20 vector containing the Pro-deleted HIV-1 sequence pHXB ⁇ Pro including the Apa ⁇ site in the vector cloning site;
  • (B) shows pHXB ⁇ ProA made by removing the Apa ⁇ site from pHXB ⁇ Pro; (C) shows pHXB ⁇ ProA made by removing the Apa ⁇ site from pHXB ⁇ Pro;
  • (D) shows plasmid pHXB ⁇ CSPro made by removal of a 200 base pair fragment encompassing the p7/p1 and p1/p6 CSs from pHXB ⁇ ProA;
  • (E) shows plasmid pHXB ⁇ CSPRTA made by extension of the deletion in pHXB ⁇ CSPro to include RT up to codon 232;
  • (F) shows plasmid pHXB ⁇ CSPRTC in which the deletion in pHXB ⁇ CSPro is extended up to RT codon 483;
  • (A) shows diagrammatically regions of the HIV-1 Gag and Gag-Pol polyproteins, with the locations of the Pro CSs indicated with a ⁇ (TF:- transframe protein;
  • NC - nucleocapsid protein
  • (B) shows the locations of the primers used in sequencing and in the generation of PCR products for assay, with their 5'->3' orientations indicated by arrows;
  • (C) shows diagrammatically by double pointed arrows in parts a) and c)-e) the regions of the HIV-1 genome that are deleted in the RVA plasmids shown in
  • FIG. 3. shows a diagrammatic summary of the competitive RVA experiments of Example 3.
  • Example 1 Construction of CS-deleted HIV-1 provirus clones for use in the RVA.
  • RVA plasmids with deletions in Pro, RT and the p7/p1 and p1/p6 CSs by extending the deletion in the existing Pro-deleted HIV-1 proviral clone (Maschera et al., 1995).
  • the Pro deletion (Fig. 1A) was extended to include the CSs (Fig. 1 C) and this CS and Pro-deleted construct was then modified using current RT-deleted constructs to include deletions in RT (Figs. 1 D and 1 E).
  • the CSs are located in a 200 bp region between the Apa ⁇ site in gag and the BstEW site at the deletion in pHXB ⁇ Pro. We deleted the CSs by removing this fragment.
  • the proviral clone pHXB ⁇ Pro comprises Pro-deleted WT HIV-1 virus HXB2 cloned into the plBI20 vector.
  • the provirus has a further Apa ⁇ site at the cloning region of the plBI20 vector. Before the 200bp Apa ⁇ -BstE ⁇ fragment containing the CSs could be removed, first it was necessary to remove this additional Apa ⁇ site.
  • Plasmid pHXB ⁇ Pro was digested with Mlu ⁇ and Xba ⁇ to remove a 29 bp fragment encompassing the / ⁇ pal site and blunt-ended with T4 DNA polymerase (New England Biolabs). The blunt ends were ligated together with an Xba ⁇ linker
  • Plasmid pHXB ⁇ CSPRTA (Fig. 1 D) contains a deletion in the CSs, Pro and RT up to codon 232 and was constructed by replacing the 5.9 kbp Hpal / BamHI fragment from pHXB ⁇ CSPro with the 5.2 kbp Bstl 1071 / BamHI fragment from the plasmid pHIVDRTBs-11071 which has a deletion in RT from codon 39 to 232 (Goulden).
  • Plasmid pHXB ⁇ CSPRTC (Fig. 1E) contains a deletion in the CSs, Pro and RT up to codon 483 and was constructed by replacing the 5.9 kbp
  • the three new RVA constructs were used for co-transfection experiments to ensure that they would allow for detection of drug resistant virus in a sample by production of viruses with drug sensitivity phenotypes consistent with the input PCR products and the regions deleted in the plasmids.
  • Co-electroporations were performed with PCR products derived from an amprenavir-resistant mutant with three Pro mutations, M46I, I47V and I50V, created by site-directed mutagenesis (see below); a lamivudine-resistant mutant with a single M184V mutation in RT created by site-directed mutagenesis (Tisdale et al., 1993); and HXB2 WT virus.
  • Clones were also isolated from a subject, Subject B, who had failed amprenavir therapy and acquired an L to F mutation at the P1' position (LP1'F) of the p1/p6 CS, which is a mutation also observed after in vitro selection of resistance with the protease inhibitors BILA 1906 BS or BILA 2185 BS (Doyon et al., 1996), during indinavir or saquinavir therapy (Zhang et al., 1997; Mammano et al., 1998) and with ABT- 378 in vitro (Carrillo et al., 1998). Clone B1 had 115V, E34G, M36I, S37E, I50V and L63P amino acid differences from the consensus subtype B Pro sequence, in addition to the LP1'F p1/p6 CS mutation.
  • the amprenavir-resistant M46I/I47V/I50V Pro mutant was created by mutagenesis of the M13 clone mpRT1/H (Larder et al. 1989) with a single synthetic oligonucleotide as described (Zoller et al., 1982; Kunkel, 1985), followed by co-electroporation into MT4 cells with an RT-deleted cloned HXB2 provirus.
  • Infected MT4 cell DNA was used as the template to generate PCR products of the amprenavir-resistant M46I/I47V/I50V Pro mutant, the lamivudine-resistant M184V RT mutant and WT HXB2.
  • Cellular DNA was purified from infected MT4 cell pellets by incubating for 16 hours at 37° in 25 mM Tris pH 7.5, 5 mM EDTA,
  • Plasma viral RNA was prepared using the Roche Amplicor HIV-1 Monitor test kit, according to the manufacturer's instructions (Mulder et al., 1994).
  • Primers used to generate RT-PCR products for cloning were RVA5' and RVA3' for the first round, and CS1 and dRTC3' for the nested reaction (table 1 and Fig 2B). Products were cloned using the TOPO TA cloning kit (Invitrogen). PCR products that cover the CSs and Pro gene, for co-transfection with pHXB ⁇ CSPro, were generated with primers CS2 and CP2 (table land Fig 2B).
  • RVAs using pHXB ⁇ Pro and a PCR product from clones A1 or B1 which covered the Pro gene but not the CSs (generated with -CS [outer] and CP2) were also carried out to create viruses with identical Pro mutations to that derived from pHXB ⁇ CSPro but without the CS mutations.
  • Primers for the generation of PCR products for RVA with pHXB ⁇ CSPRTA were CS2 and dRTA3'.
  • the primers used were CS2 and IN3'.
  • RT-PCR of RNA from plasma was carried out as follows: The first round PCR consisted of two layers of reagents in a single tube, separated by a wax layer (Ampliwax PCR Gem 100, Perkin Elmer). The upper layer contained the reagents for reverse transcription in a total volume of 50 ⁇ l and consisted of 50 mM Tris-HCI (pH 8.3), 75 mM KCI, 3 mM MgCI 2 , 10 mM DTT, 5% (v/v) DMSO, 500 ⁇ M each dNTP, 20 ⁇ g/ml BSA, 250 ng 3 ' primer, 40 u Rnasin ribonuclease inhibitor (Promega), 200 u Superscript II RT (Gibco BRL) and 25 ⁇ l RNA template.
  • the lower layer contained (in a total volume of 50 ⁇ l) 1 mM Tris (pH 8.0), 0.1 mM EDTA, 5% (v/v) DMSO, 250 ng 5' primer and 2.5 u AmpliTaq DNA Polymerase (Perkin Elmer).
  • Second round PCR reactions and amplifications from infected MT4-cell DNA or plasmid clones consisted of (in a total volume of 100 ⁇ l) 20 mM Tris (pH 8.8), 25 mM KCI, 1.5 mM MgCI 2) 5% (v/v) DMSO, 200 ⁇ M each dNTP, 250 ng of each primer and 5 ⁇ l template DNA.
  • Thermal cycling was carried out in a Perkin Elmer GeneAmp PCR system 9600 with the following cycles: 45° for 45 minutes (first round only); 95° for 20 seconds, 55° for 10 seconds, 72° for 60 seconds (5 cycles); 90° for 10 seconds, 55° for 10 seconds, 72° for 60 seconds + 5 extra seconds for each consecutive cycle (30 cycles).
  • RVA plasmids were linearised by digestion with a restriction enzyme that cut at the site of the deletion. Plasmids pHXB ⁇ Pro, pHXB ⁇ CSPro and pHXB ⁇ CSPRTC were cut with BstEW, pHXB ⁇ CSPRTA was cut with >Apal. Transfection of MT4-cells was based on the method described by Kellam and Larder (1994). Briefly, 10 ⁇ g of linearised plasmid was electroporated into MT4 cells with approximately 5 ⁇ g of the PCR product, which had been purified with the QIAquick Spin PCR Purification Kit (Qiagen).
  • IC50 concentration of drug required to increase the absorbency to levels 50% of those in uninfected control cells
  • Plasmids pHXB ⁇ CSPR and pHXB ⁇ CSPRTA produced APV-resistant virus when co-transfected with PCR products derived from the M46I/I47V/I50V PR mutant (25 FR); whilst virus derived from the M184V RT mutant showed APV sensitivity comparable to HXB2.
  • the constructs therefore appear to produce viruses with a PR phenotype expected from the input PCR product.
  • the RT phenotype of viruses produced in the RVA were also consistent with the properties of the input PCR products and the region of deletion in the plasmids, as demonstrated by the acquisition of lamivudine resistance when pHXB ⁇ CSPRTA or pHXB ⁇ CSPRTC (>18 FR), but not pHXB ⁇ CSPR was co- transfected with PCR products from the M184V RT mutant.
  • One of the cloned CS mutants was from a subject designated Subject A, who had received indinavir (IDV) therapy but viral load data had indicated therapy failure (previous therapy had also included zidovudine, lamivudine and stavudine).
  • Sequencing of plasma virus from Subject A revealed an A to V mutation at the P 2 position (AP V) of the p7/p1 CS, a mutation observed previously in subjects receiving IDV therapy (Zhang et al., 1997).
  • Clone A1 had the AP 2 V p7/p1 CS mutation and also 115V, I54V, R57K, I62V, L63P, H69Y, A71T, I72E, V82A and I85V differences from the consensus
  • Subtype B PR sequence Clones were isolated from a second subject, Subject B, who had failed amprenavir (APV; VX-478; 141W94) therapy (therapy also included zidovudine and lamivudine).
  • Plasma virus from Subject B had acquired an L to F mutation at the P-T position (LP-i'F) of the p1/p6 CS, which is a mutation also observed after in vitro selection of resistance with the protease inhibitors BILA 1906 BS or BILA 2185 BS (Doyon et al., 1996), during IDV or saquinavir therapy (Zhang et al., 1997; Mammano et al., 1998) and with ABT-378 in vitro (Carrillo et al., 1998).
  • Clone B1 had 115V, E34G, M36I, S37E, I50V and L63P amino acid differences from the consensus Subtype B PR sequence, in addition to the LPi'F p
  • pHXB ⁇ PR and pHXB ⁇ CSPR produced viruses with similar IDV susceptibility (8.1 and 8.6 FR respectively) with PCR products derived from clone A1 (Table 2). Similar results were seen with viruses derived from clone B1 when they were assessed for APV resistance with the two constructs (2.5 and 2.6 FR respectively). Thus the presence or absence of the CS mutations did not significantly affect the drug susceptibility phenotype of RVA products generated from a homogeneous template. This is consistent with the original data of Doyon et al. (1996), which suggested an effect of CS mutations on viral fitness rather than directly on drug susceptibility.
  • virus B1 appeared to have increased susceptibility to IDV (>2.4 fold increased susceptibility in both recombinants, the actual values were out of the range of this experiment). Small increases in cross-susceptibility of APV-resistant viruses with other Pis have been documented previously (Tisdale, 1996).
  • CSs When the CSs are not included in the PCR product used for the RVA, one would predict that when CS mutations were present in the source plasma virus population, growth of the resistant viruses may be impaired and viruses with fewer mutations and hence less resistance could have a growth advantage. Selection of less resistant viruses in the RVA as a result of a growth advantage may lead to an inaccurate determination of resistance and cross resistance to Pis in subsequent analyses, possibly impacting decisions made about therapy regimens.
  • the selection process that might occur in the RVA during the growth of a heterogeneous population of recombinant viruses derived from plasma was simulated by mixing known proportions of input PCR products from a CS mutation harbouring Pl-resistant virus with those from WT virus (Fig. 3). Molecular clones were used as PCR templates to enable the ratio of mutant : WT to be precisely controlled.
  • CSs and Pro gene were made from the plasma virus of 2 subjects infected with viruses containing CS mutations.
  • the Pro gene, or CSs and Pro gene were amplified from the clones by PCR and mixed with WT products in ratios of mutant to WT of 4:1 or 9:1.
  • the Pro-only products were co- electroporated into MT4 cells with pHXB ⁇ Pro and the CS+Pro products were co- electroporated with pHXB ⁇ CSPro.
  • Relative proportions of mutant and WT in the resulting recombinant virus populations were determined by assessing their susceptibility to indinavir or amprenavir and sequencing cloned PCR products derived from the infected cell pellets.
  • PCR products that covered the CSs and Pro gene were generated from clone A1 using primers CS2 and CP2. Products covering only the Pro gene were generated with primers -CS (outer) and CP2. Corresponding WT products were generated using plasmid pHIV ⁇ Bs-EII (Kellam and Larder, 1994) as the template
  • the concentration of the purified PCR products were determined from the absorbency at 260 nm or by visualising in agarose gels, and the products from clone A1 were mixed with WT in ratios of 4:1 or 9:1 (mutant:WT). Approximately 5 ⁇ g of the mixtures were coelectroporated into MT4 cells with 5 ⁇ g of linearised pHXB ⁇ CSPro (for products which included the CSs) or pHXB ⁇ Pro (products which did not include the CSs). Co-electroporations of A1 PCR DNA alone with the RVA constructs were also performed to generate A1 -derived viruses with or without the CS mutation.
  • RVA5' and Comb3 The PCR products were cloned and 11 or 12 clones from each RVA were sequenced. The resulting susceptibility and sequence data are presented in table 3.
  • the virus reconstructed from clone A1 without the CS mutation had indinavir sensitivity similar to the reconstructed virus with the CS mutation (A1 +CS) when they were generated from a homogeneous PCR product (table 3, experiment A).
  • A1-CS PCR products were mixed with WT-CS products in ratios of 4:1 or 9:1 , the resulting RVA virus mixtures were only marginally more resistant to IDV than WT virus.
  • all 11 or 12 cloned PCR products derived from the cell DNA were WT, thus WT virus had out-competed A1-CS and given an RVA product unrepresentative of the input PCR products.
  • the growth rates of the virus clones were compared with that of a WT virus that was reconstructed from pHXB ⁇ CSPro.
  • Duplicate cultures of 1 x 10 6 MT4 cells were infected with an amount of virus stock containing 10 ng of p24 for each of the PI resistant viruses or with a clonal WT virus derived from pHXB ⁇ CSPR.
  • the infected cells were incubated in 10 ml of growth medium and 0.5 ml aliquots of the supematants were taken at 2, 4, 6 and 8 days post-infection-
  • the p24 concentrations in each aliquot were then determined in order to assess the relative virus growth rates.
  • Concentrations of HIV-1 p24 in tissue culture supematants or virus stocks were determined with the Murex HIV Antigen Mab kit (Abbott Diagnostics). The data is presented in figure 4.
  • Pl-resistance mutations lead to multiple Gag and Gag-Pol cleavage defects (Doyon et al., 1996; Maschera et al., 1996a, b; Mammano et al., 1998; Zennou et al., 1998).
  • mutations occur at CSs other than p7/p1 and p1/p6 should be considered when examining resistance to Pro inhibitors in the RVA, since sites outside the input PCR product would select against resistant virus growth. Nevertheless the p7/p1 and p1/p6 sites appear to be the preferential sites of mutation in response to the reduction of viral fitness.

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Abstract

La présente invention concerne des méthodes de génération de virus recombinants à partir d'échantillons, tels que des échantillons de virus non caractérisés ou des échantillons cliniques, l'utilisation dans des analyses des virus ainsi générés, principalement en vue de détecter une sensibilité virale altérée à des médicaments antiviraux. L'invention concerne également des réactifs, et plus particulièrement des constructions de vecteurs d'acide désoxyribonucléique (ADN), destinés à être utilisés dans ces méthodes et analyses. Les analyses sont conçues pour une détection de virus résistants plus précise et sensible que les analyses connues, notamment par la prise en compte des mutation compensatoires intervenant dans des séquences nucléotidiques autres que celles codant pour la cible du médicament antiviral.
PCT/GB2000/001639 1999-04-28 2000-04-28 Analyse amelioree et ses reactifs WO2000066774A2 (fr)

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EP00927464A EP1226270A2 (fr) 1999-04-28 2000-04-28 Analyse amelioree et ses reactifs
JP2000615396A JP2002542804A (ja) 1999-04-28 2000-04-28 改良されたアッセイとその試薬

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Cited By (8)

* Cited by examiner, † Cited by third party
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WO2001079542A3 (fr) * 2000-04-14 2002-06-06 Glaxo Group Ltd Essai de phenotypage et reactifs y relatifs
FR2829503A1 (fr) * 2001-03-23 2003-03-14 Bioalliance Pharma Nouvelee methode d'analyse des caracteristiques phenotypiques des virus de l'immunodeficience humaine(vih)
FR2829506A1 (fr) * 2001-03-23 2003-03-14 Bioalliance Pharma Nouvelle methode d'analyse des caracteristiques phenotypiques des virus de l'immunodeficience humaine(hiv)
FR2829502A1 (fr) * 2001-03-23 2003-03-14 Bioalliance Pharma Nouvelle methode d'analyse des caracteristiques phenotypiques des virus de l'immunodeficience humaine(vih)
FR2829501A1 (fr) * 2001-03-23 2003-03-14 Bioalliance Pharma Nouvelle methode d'analyse des caracteristiques phenotypiques des virus de l'immunodeficience humaine(vih)
WO2002038792A3 (fr) * 2000-11-10 2003-09-25 Bioalliance Pharma Nouvelle methode d'analyse des caracteristiques phenotypiques des virus de l'immunodeficience humaine (vih)
US7306901B2 (en) 2001-08-08 2007-12-11 Tibotec Pharmaceuticals, Ltd. Methods and means for assessing HIV envelope inhibitor therapy
EP2836610A4 (fr) * 2012-04-12 2015-12-30 Zeus Scientific Inc Procédés de mesure de l'activité de la polymérase pouvant être utilisés en vue de mesures quantitatives sensibles d'une quelconque activité d'extension par la polymérase et pour déterminer la présence de cellules viables

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WO2007080874A1 (fr) * 2006-01-10 2007-07-19 Meiji Seika Kaisha, Ltd. Fragment genetique mutant de staphylocoque faiblement sensible à un glycopeptide et son usage

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WO2000073511A1 (fr) * 1999-05-28 2000-12-07 Virco Nv Nouveaux profils mutationnels dans la transcriptase inverse du vih-1 en relation avec une pharmacoresistance phenotypique

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001079542A3 (fr) * 2000-04-14 2002-06-06 Glaxo Group Ltd Essai de phenotypage et reactifs y relatifs
WO2002038792A3 (fr) * 2000-11-10 2003-09-25 Bioalliance Pharma Nouvelle methode d'analyse des caracteristiques phenotypiques des virus de l'immunodeficience humaine (vih)
FR2829503A1 (fr) * 2001-03-23 2003-03-14 Bioalliance Pharma Nouvelee methode d'analyse des caracteristiques phenotypiques des virus de l'immunodeficience humaine(vih)
FR2829506A1 (fr) * 2001-03-23 2003-03-14 Bioalliance Pharma Nouvelle methode d'analyse des caracteristiques phenotypiques des virus de l'immunodeficience humaine(hiv)
FR2829505A1 (fr) * 2001-03-23 2003-03-14 Bioalliance Pharma Nouvelle methode d'analyse des caracteristiques phenotypiques des virus de l'immunodeficience humaine(vih)
FR2829502A1 (fr) * 2001-03-23 2003-03-14 Bioalliance Pharma Nouvelle methode d'analyse des caracteristiques phenotypiques des virus de l'immunodeficience humaine(vih)
FR2829501A1 (fr) * 2001-03-23 2003-03-14 Bioalliance Pharma Nouvelle methode d'analyse des caracteristiques phenotypiques des virus de l'immunodeficience humaine(vih)
FR2829504A1 (fr) * 2001-03-23 2003-03-14 Bioalliance Pharma Nouvelle methode d'analyse des caracteistiques phenotypiques des virus de l'immunodeficience humaine(vih)
US7306901B2 (en) 2001-08-08 2007-12-11 Tibotec Pharmaceuticals, Ltd. Methods and means for assessing HIV envelope inhibitor therapy
EP2836610A4 (fr) * 2012-04-12 2015-12-30 Zeus Scientific Inc Procédés de mesure de l'activité de la polymérase pouvant être utilisés en vue de mesures quantitatives sensibles d'une quelconque activité d'extension par la polymérase et pour déterminer la présence de cellules viables

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AU4586700A (en) 2000-11-17
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JP2002542804A (ja) 2002-12-17
EP1226270A2 (fr) 2002-07-31

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