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Selection of Antiretroviral Resistance in the Latent Reservoir of Human Immunodeficiency Virus Type 1 during Successful Therapy J. MARTINEZ-PICADO1,2, M. P. DePASQUALE1, N. A. KARTSONIS1, G. HANNA1, J. WONG3, D. FINZI4, E. ROSENBERG1, H. GUNTHARD3, L. SUTTON1, A. SAVARA1, C. PETROPOULOS5, N. HELLMANN5, B. D. WALKER1, D. D. RICHMAN3, R. SILICIANO4, and R.T. D’AQUILA1*.
1Massachusetts Gen. Hosp. & Harvard Med. Sch., Boston, MA; 2Fndn. irsiCaixa, Barcelona, Spain; 3Univ. of California San Diego and VA Med. Ctr., La Jolla, CA; 4Johns Hopkins Univ., Baltimore, MD; and 5ViroLogic Inc., South San Francisco, CA Introduction: Latent integrated HIV-1 can be reactivated in vitro from resting memory CD4+ T lymphocytes. Low level virus replication may continue in vivo even when plasma HIV-1 RNA is <50 copies/ml. Many patients have transient episodes of RNA >50 copies/ml. However, initial studies did not identify new drug resistance mutations in replication-competent HIV recovered in vitro from the blood latent reservoir during successful therapy (Wong, et al. & Finzi et al., Science 1997).
Methods: Viruses were isolated from PBMC during therapy which suppressed plasma HIV RNA to <50 copies/ml from 12 patients: 6 patients’ viruses were biologically cloned (Finzi, et al.) and 6 pools of viruses isolated by Wong, et al. were molecularly cloned. Pol genotyping was by cycle sequencing and LiPA. Env C2-V3 was sequenced from biologic clones. Phylogenetic analyses (PHYLIP) and recombinant virus drug susceptibility assays (ViroLogic) were done.
Results: Clonal sequencing analyses of replication-competent viruses recovered in vitro from latently infected blood lymphocytes identified new drug resistance mutations selected during successful therapy in 4 of the 5 drug-experienced, established infection patients with episodes of transient viremia. New resistance mutations did not develop in the 7 patients who did not have transient viremia. New resistance mutations were added to pre-existing mutational patterns in both protease (PR) and reverse transcriptase (RT) (two patients), only PR (one patient), or only RT (one patient). In one of these patients studied over 6 time points during successful therapy, PI-selected mutations accumulated in a virus sub-population and increasing resistance was confirmed by susceptibility assay. Phylogenetic analyses also showed drug selection pressure on protease, but not env. Repeated sampling over time, consistent findings in different patients, association with evidence of ongoing replication (env genetic diversity and transient viremia), and the specific mutation patterns observed suggest that the new resistance mutations were indeed selected during therapy.
Conclusion: Drug resistant HIV-1 can be selected in vivo in the absence of sustained rebound of viremia during treatment of established infection with regimens currently considered optimal.
Key Words: genotyping, latent reservoir, resistance
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