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1st International Workshop on HIV Drug Resistance & Treatment Strategies25-28 June 1997, St. Petersburg, Florida, USA |
GENOTYPIC OR PHENOTYPIC SUSCEPTIBILITY TESTING MAY NOT PREDICT CLINICAL RESPONSES TO INDINAVIR
Antiviral Therapy 1997;2 (Suppl 5):31 (abstract no. 47)
JH Condra, DJ Holder, DJ Graham, M Shivaprakash, DT Laird, WA Schleif, JA Chodakewitz and EA Emini
Merck Research Laboratories, West Point, PA 19486, USA
Resistance to indinavir results from the accumulation of multiple amino acid substitutions in the HIV-1 protease, potentially involving 11 or more residues of the protease. Their contributions to resistance, though, are dependent upon the genetic contexts in which they appear.
We have examined the appearance of resistance-associated amino acid substitutions with respect to the timing of vRNA rebounds in patients. The appearance of these mutations sometimes preceded measurable viral phenotypic changes. In two patients, new substitutions L10R/M46I or L10I/K20M/V82A were observed, though the viruses were still phenotypically wild-type (indinavir CIC95 100 nM). Nonetheless, these viral populations prevailed during RNA rebounds. Thus, although resistance mutations were detected, the phenotypic assay was incapable of measuring levels of resistance that were associated with breakthrough.
We previously reported that in a patient receiving indinavir who discontinued therapy, the resistant viral population was rapidly replaced by indinavir-susceptible viruses [1]. Moreover, after 34 weeks in the absence of indinavir, viruses that were identical to the previously observed resistant viruses replaced that population once therapy was resumed. We have measured the frequency of the V82F mutation during this patient's alternating therapies. Multiple molecular clones of PCR-amplified protease genes were screened for V82F by colony hybridization with an oligonucleotide probe. Although the V82F mutation was undetectable (frequency <0.02 by end-point dilution) at week 0, its frequency rose to 1.0 at weeks 22 and 36. By week 49, 10 weeks after discontinuing indinavir, the frequency dropped to 0.04, declining to 0.0005 at week 73, when the patient resumed indinavir. At this time, viral phenotypic assays indicated full susceptibility to indinavir. At week 76, the V82F frequency increased to about 0.8, and the viral population was again phenotypically resistant.
These results indicate that the methods normally used to assess genotypic changes in the protease, either by sequencing populations or relatively small numbers of molecular clones, were inadequate to detect the low frequencies of mutations that were present and eventually founded resistant populations. These methods are generally unable to detect mutants comprising less than 10 to 25% of mixed populations. This pre-existing resistance was also undetectable phenotypically. Thus, standard viral genotypic and phenotypic assays may not adequately predict clinical responses to indinavir. The complexity of HIV viral populations and the insensitivities of available assays may thus limit the ability of in vitro techniques to predict clinical responses to antiviral therapy.
1. Condra JH, Schleif WA, Blahy OM, Gabryelski LJ, Graham DJ, Quintero JC, Rhodes A, Robbins HL, Roth E, Shivaprakash M, Titus D, Yang T, Zhang J, Deutsch PJ, Squires KE & Emini EA. Dynamics of HIV replication in relation to drug resistance. 4th International Workshop on HIV Drug Resistance. Kauai, Hawaii, USA 1995.
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1997-06-25
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