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Session 75 Poster Session Resistance to Antiretroviral Chemotherapeutic Agents Session Time: 4:30-6:30 pm Room 4E-F

555-T. Co-Existence and Co-Evolution of Viral Populations with Distinct Genotypes in Patients Failing Treatment with Protease Inhibitors C. Charpentier, D. E. Dwyer, D. Lecossier, F. Clavel, and A. J. Hance* INSERM U552, Paris, France Background: The acquisiton of viral resistance to protease inhibitors results from the accumulation of resistance mutations, but little data are available concerning the evolutionary pathway(s) followed during this process. Methods: We evaluated 7 patients failing treatment with protease inhibitors in whom a resistance genotype containing the L90M mutation developed. To investigate the kinetics of appearance of L90M, the proportion of viral sequences containing L90M was evaluated in all archived serum samples using selective amplification of mutated sequences by real-time PCR (an approach that can detect minority populations containing L90M that represent as little as 0.1% of total virus). At various times during the evolution of resistance in two patients, viral protease sequences present in plasma were cloned and screened by selective PCR for the presence of the L90M mutation. Clones representative of the populations with and without the L90M mutation were sequenced, permitting reconstruction of the selection events occurring during the development of resistance. Results: For 3 patients, the L90M mutation was present in the majority of circulating viruses within 3 months of treatment failure. In the remaining 4 patients, minority populations expressing L90M were detectable for 10-35 months prior to the establishment of a majority population containing this mutation. Analysis of clones in 2 of these patients explained the origin of these minority populations. In both cases, early resistance was associated with the emergence of a single genotype containing few resistance mutations that gave rise to all subsequent variants. On this background, L90M and several other mutations were added in various combinations, resulting in the coexistence of viral populations with several distinct genotypes. The relative abundance of a given quasi-species varied considerably over time, and viruses in each lineage continued to evolve. The L90M mutation (and several other resistance mutations) were found to occur independently on 2 or more occasions during the evolution of resistance. Conclusions: After an initial bottleneck followed by extensive diversification, viral resistance to PIs is characterized by the coexistence and coevolution of viral populations expressing several distinct genotypes.

©2002 9th Conference on Retroviruses and Opportunistic Infections