293   Characterization of Minority Viral Populations Expressing Protease Resistance Mutations in Patients Undergoing Structured Treatment Interruptions (STI).

A. Hance*¹, V. Lemiale¹, J. Izopet², D. Lecossier¹, V. Joly¹, P. Massip², F. Mammano¹, D. Descamps¹, F. Brun-Vezinet¹, and F. Clavel¹.
¹CRIV Bichat-Claude Bernard, Paris and²CHU Purpan, Toulouse, France.

Background:Following STI in HIV-1-infected patients harboring drug-resistant virus, resistance mutations are no longer detectable in plasma virus by genotyping in most patients. We have recently shown that minority resistant populations (1—10% of total virus) can be detected in many patients using a real-time-PCR-based method permitting the selective amplification and quantification of viral populations carrying key protease mutations. The origin of these resistant quasi-species has not been clearly defined.

Methods:To genotype minority populations in plasma samples obtained 5 mo (patient 1) or 3 mo (patient 2) after STI, most of the protease gene was amplified by nested RT-PCR using primers that amplify both mutant and wild-type virus and techniques that prevent recombination during PCR. PCR products were cloned, and clones with V82A or L90M resistance mutations were identified.

Results:Clones containing resistance mutations were identified in proportions consistent with the results obtained by sequence-selective PCR (patient 1, 3% V82A by PCR and 3/176 clones; patient 2, 4% L90M by PCR and 5/131 clones). Clones were not homogeneous in either patient, and most of the viruses had a genotype that was intermediate between highly resistant pre-STI virus and majority wild-type virus that emerged after STI. In patient 1, clones containing 2, 3 and 4 of the 5 mutations present prior to STI were identified and appeared to recapitulate the steps leading to the development of full protease resistance. No evidence for recombination was found, and reversion of mutations could not explain the appearance of most of the partially resistant strains.

Conclusions:Patients failing treatment with protease inhibitors continue to harbor viruses selected at earlier stages of drug resistance that proliferate better under drug-free conditions than fully drug-resistant strains present prior to STI. Thus, optimal post-STI treatment regimens must prevent the emergence of previously selected resistant populations, including those with genotypes not detected immediately before stopping therapy.