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Antiviral Agents

Emergence of Resistant Human Immunodeficiency Virus Type 1 in Patients Receiving Fusion Inhibitor (T-20) Monotherapy

Xiping Wei, Julie M. Decker, Hongmei Liu, Zee Zhang, Ramin B. Arani, J. Michael Kilby, Michael S. Saag, Xiaoyun Wu, George M. Shaw, John C. Kappes
Xiping Wei
1Howard Hughes Medical Institute, Departments of
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Julie M. Decker
1Howard Hughes Medical Institute, Departments of
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Hongmei Liu
2 Medicine
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Zee Zhang
2 Medicine
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Ramin B. Arani
3Biostatistics, University of Alabama at Birmingham, Birmingham, Alabama 35294
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J. Michael Kilby
2 Medicine
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Michael S. Saag
2 Medicine
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Xiaoyun Wu
2 Medicine
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George M. Shaw
1Howard Hughes Medical Institute, Departments of
2 Medicine
4Microbiology
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John C. Kappes
2 Medicine
3Biostatistics, University of Alabama at Birmingham, Birmingham, Alabama 35294
5Birmingham Veterans Affairs Medical Center, Research Service, Birmingham, Alabama 35233
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  • For correspondence: kappesjc@uab.edu
DOI: 10.1128/AAC.46.6.1896-1905.2002
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  • FIG. 1.
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    FIG. 1.

    HIV-1 entry assay. (A) Illustration of lentivirus vector used to transduce JC53 cells (LTR, long terminal repeat; SD, splice donor; Ψ, RNA packaging signal; RRE, rev-responsive element; IRES, internal ribosomal entry site). Infectious stocks of vector containing either β-Gal or lucif were prepared by transfection of 293T cells as described previously (14) and used to transduce JC53 cells. (B) Analysis of viral infectious units and lucif activity in JC53BL-13 cells. Serial twofold dilutions of the T-tropic NL4-3 and M-tropic YU-2 viruses (1, 11), beginning at 5,120 infectious units, as determined by the β-Gal assay, were used to infect (in quadruplicate) JC53BL-13 cells plated in a 96-well tissue culture plate. Forty-eight hours after infection, the replica wells were lysed, pooled, and analyzed for lucif activity by using an automated Lumistar XL system (BMG Lab Technologies). lucif activity was plotted against the number of infectious units. (C) Phenotypic analysis of the HIV-1 env clones for susceptibility to inhibition by T-20. Full-length env genes were cloned into the pCDNA3.1 eukaryotic expression plasmid. Individual pCDNA3.1-env clones were cotransfected with pSG3Δenv into 293T cells. Pseudotyped progeny virions collected from supernatants of the transfected cells were used to infect JC53BL-13 cells in either the absence or the presence of various concentrations of T-20. Virus infection induces expression of the lucif reporter gene, which was quantified to measure virus entry.

  • FIG. 2.
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    FIG. 2.

    Population sequence analysis of plasma HIV-1 env. (A) Comparison of the FP, HR1, and HR2 amino acid sequences pre- and posttreatment with T-20. The plasma virus from one subject (3-1) in the 3-mg dose group, two subjects (10-2 and 10-3) in the 10-mg dose group, and four subjects each in the 30- and 100-mg groups were analyzed by population sequencing from before day 0 (d 0) and after 14 days of treatment (d14) with T-20. The sequences were analyzed using Sequencher software and aligned against the LAI consensus sequence. The FP, HR1, and HR2 regions are depicted. The region within HR2 to which T-20 binds is underlined. (B) Quantitative detection of mutant plasma virus by automated population DNA sequencing. DNA sequence chromatograms of specified regions within HR1 are shown for patients 30-1 (residues 36 to 38), 30-2 (residues 45 to 47), and 30-3 (residues 68 to 69). Chromatograms are shown for each subject from before day 0 and after 14 days (Day 14) of T-20 treatment. The sequences shown were obtained from, and therefore are presented as, the minus (noncoding) DNA strand. The minus-strand sequence corresponds to the plus strand (coding), which is indicated on each chromatogram. Codon changes are indicated as plus-strand substitutions.

  • FIG. 3.
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    FIG. 3.

    Sequence analysis of env clones derived from plasma virus. Prior to (d0) and 14 days after (d14) treatment with T-20, the full-length env gene was amplified from viral RNA by RT-PCR and cloned into pCDNA3.1. The sequences of multiple clones derived from subjects 30-1 (A) and 30-3 (B) were analyzed using Sequencher software and aligned against the LAI consensus sequence. The sequence of the entire HR1 domain is illustrated. The vertical shaded rectangles highlight the GIV motif.

  • FIG. 4.
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    FIG. 4.

    Sensitivity of GIV mutant Env to inhibition by T-20. One thousand infectious units of HIV-1 pseudotyped with mutant or wt Envs derived from patient 30-1 was used to infect cultures of JC53BL cells containing 0, 0.04, 0.2, 1, 5, and 25 μg of T-20 per ml. Two days later, virus entry was measured by analyzing the indicator cells for lucif activity. Virus infectivity was calculated by dividing the mean lucif activity value at each of the drug concentrations by the mean value of the control (0 mg). Resistance to T-20 is depicted by plotting relative infectivity on the y axis against T-20 concentration on the x axis. (A) HIV-1/SG3Δenv was pseudotyped with the G36D mutant Envs 57 and 2, the V38A mutant Envs 18 and 21, and wt Envs (wt-d0 and wt-d14). (B) Sequence analysis of the HR2 and V3 Env domains for the wt (d0-wt), no. 2 (d14-02), and no. 57 (d14-57) env clones. (C) HIV-1/SG3Δenv stocks were pseudotyped with G36D mutant Env 57, the 32H/36D double mutant Envs (32H, 36D), the 32R/36D double mutant Env (32R, 36D), and the wt Env (wt-d0). The results depicted in panels A and B were highly reproducible and are representative of three independent experiments.

  • FIG. 5.
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    FIG. 5.

    Sensitivity of GIV mutant Env to inhibition by T-20. HIV-1 pseudotyped with mutant or wt env derived from patient 30-3 was used to infect cultures of JC53BL cells exactly as described for Fig. 4. Pseudotyped HIV-1/SG3Δenv stocks were prepared with the V38M and I37V mutant and wt (wt-d0) Envs. The results are representative of three independent experiments.

  • FIG. 6.
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    FIG. 6.

    Analysis of T-20 resistance from cultured plasma virus. One thousand infectious units of HIV-1 derived by the coculture of plasma from patient 30-1, prior to (d-0) and after 14 days (d-14) of T-20 treatment, was used to infect JC53BL indicator cells in either the absence or presence of different concentrations (0, 0.04, 0.2, 1, 5, and 25 μg/ml) of T-20. Two days later, the indicator cells were lysed and analyzed for lucif activity. These results are representative of two independent experiments. The DNA sequence chromatogram specific for the GIV sequence is shown (upper right) for the day 14 plasma virus that was isolated after 18 days of cocultivation with normal-donor PBMC.

Tables

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  • TABLE 1.

    Comparison of sensitivities of JC53BL cells and PBMC to HIV-1 infection

    ELISA result for and phenotype of primary HIV-1 isolateaPBMCbJC53BLc
    Namep24 (pg/ml)PhenotypeTCID50p24:TCID50TCID50p24:TCID50
        K19.5 × 105NSI1.8 × 1055.32.3 × 1054.1
        K23.2 × 105SI4.6 × 1046.91.2 × 1052.7
        K34.2 × 105SI1.8 × 1052.34.5 × 1050.9
        K45.5 × 105NSI4.6 × 10411.99.5 × 1050.58
        K56.7 × 105SI4.6 × 10414.61.8 × 1053.7
        K68.2 × 105SI4.6 × 10417.88.5 × 1049.6
        K71.3 × 105SI1.2 × 10410.84.5 × 1042.9
        K86.8 × 105NSI4.6 × 10414.81.1 × 1056.2
        K94.2 × 105NSI4.6 × 1049.11.3 × 1053.2
        K105.3 × 105SI4.6 × 10411.51.1 × 1054.8
        K111.2 × 105SI1.8 × 1050.74.1 × 1050.29
        K125.4 × 105NSI4.6 × 10411.79.0 × 1046.0
        K136.5 × 104SI1.2 × 1045.43.8 × 1041.7
        K145.0 × 105NSI4.6 × 10410.92.1 × 1052.4
        K152.1 × 105NSI4.6 × 1044.61.2 × 1051.8
        K162.2 × 105NSI4.6 × 1044.83.6 × 1050.61
        K172.7 × 105SI1.2 × 10422.51.0 × 1052.7
        K182.6 × 105NSI4.6 × 1045.72.0 × 1051.3
        K195.1 × 105NSI4.6 × 10411.12.8 × 1051.8
        K202.1 × 105SI1.2 × 10417.54.5 × 1044.7
    • ↵ a Primary virus isolates were derived by coculture (10 days) of HIV-1-infected patient PBMC with PHA-stimulated normal donor PBMC. Virus concentrations were determined by HIV-1 p24 antigen enzyme-linked immunosorbent assay (ELISA) (Beckman-Coulter), and the syncytium-inducting (SI) or non-syncytium-inducing (NSI) phenotypes were determined on MT2 cells.

    • ↵ b Analysis of PBMC-derived HIV-1 infectivity by endpoint dilution culture of PHA-stimulated normal-donor PBMC. Virus titers for 50% tissue culture infectivity doses per milliliter (TCID50/ml) were calculated using the Spearman-Karber formula.

    • ↵ c Analysis of PBMC-derived HIV-1 infectivity by counting β-Gal positive JC53BL positive cell colonies under a microscope.

  • TABLE 2.

    Plasma vRNA load in patients treated with a 30-mg dose of T-20

    DaySubjecta
    30-130-230-330-4
    05.714.254.934.50
    35.523.984.584.29
    75.264.114.223.64
    105.104.484.093.32
    145.134.764.263.73
    • ↵ a Four individuals were administered 30 mg of T-20 twice daily. The numbers represent plasma vRNA loads (log10) over the course of 14 days of treatment, as determined by bDNA (Chiron Inc.) testing.

  • TABLE 3.

    Site-directed mutagenesisa

    MutationIC50Fold change
    wt0.24
    G36D1.45.8
    G36D/Q32R1.35.4
    V38A/Q32R1.56.3
    • ↵ a Mutations were introduced into a wt, day 0 env clone (pcDNA3.1-env) derived from patient 30-1 by using the Quik-Change kit (Stratagene Inc.) and degenerate oligonucleotide primers. Mutations were identified by nucleotide sequence analysis.

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Emergence of Resistant Human Immunodeficiency Virus Type 1 in Patients Receiving Fusion Inhibitor (T-20) Monotherapy
Xiping Wei, Julie M. Decker, Hongmei Liu, Zee Zhang, Ramin B. Arani, J. Michael Kilby, Michael S. Saag, Xiaoyun Wu, George M. Shaw, John C. Kappes
Antimicrobial Agents and Chemotherapy Jun 2002, 46 (6) 1896-1905; DOI: 10.1128/AAC.46.6.1896-1905.2002

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Emergence of Resistant Human Immunodeficiency Virus Type 1 in Patients Receiving Fusion Inhibitor (T-20) Monotherapy
Xiping Wei, Julie M. Decker, Hongmei Liu, Zee Zhang, Ramin B. Arani, J. Michael Kilby, Michael S. Saag, Xiaoyun Wu, George M. Shaw, John C. Kappes
Antimicrobial Agents and Chemotherapy Jun 2002, 46 (6) 1896-1905; DOI: 10.1128/AAC.46.6.1896-1905.2002
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KEYWORDS

Anti-HIV Agents
HIV Envelope Protein gp41
HIV infections
HIV-1
Peptide Fragments

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