Background:  Latent HIV-1 DNA persists in resting memory CD4⁺ T cells despite HAART and represents a barrier to eradication of infection. Theoretically, the resting CD4⁺ T cell reservoir preserves all major forms of the virus that have circulated in a patient’s plasma, both wild type and drug resistant. A novel culture assay that allows genetic delineation of HIV-1 produced by resting CD4⁺ T cells regardless of the level of plasma viremia was used to study the persistence of non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance; these results were compared to proviral DNA sequences.  

Methods:  We studied 6 patients with previously documented NNRTI resistance, which was no longer detectable in the plasma. Samples were obtained 38 to 64 months after stopping the NNRTI at which time 3 of 6 patients had undetectable viral loads on HAART and 3 of 6 patients were viremic on partially suppressive regimens. Purified resting CD4⁺ T cells were cultured with activating stimuli for 10 days with RT and integrase inhibitors to ensure that only stably integrated latent HIV-1 was detected. HIV RT sequences from culture supernatants, plasma, and proviral DNA (for the 3 aviremic patients) were amplified by limiting-dilution polymerase chain reaction (PCR), sequenced, and neighbor-joining phylogenetic trees were generated.

Results:  Virus production from resting CD4⁺ T cells with integrated latent HIV-1 genomes was detected in 5 of 6 patients. Multiple independent HIV-1 clones with K103N or Y181C were found in the resting CD4⁺ T cells but not in the plasma of 3 of 5 patients. NNRTI resistance mutations were also present in proviral DNA sequences from 3 of 3 aviremic patients. Archiving of NNRTI-resistant virus occurred irrespective of the specific mutation (Y181C or K103N), length of NNRTI exposure (4 to 29 months), level of viremia at time of NNRTI discontinuation (3.6 to 6 log₁₀ copies/mL), or time since exposure (38 to 64 months).

Conclusions:  A novel culture assay demonstrates that drug resistant HIV-1 species are present in the latent resting CD4⁺ reservoir as detected by virus produced from the cells as well as proviral DNA sequences long after resistance is no longer detectable in the plasma. For patients with a history of NNRTI resistance, these results provide the first direct evidence that the K103N or Y181C mutation can persist in resting CD4⁺ T cells for years after discontinuation of the drug.  Archived replication competent forms of HIV-1 have the potential to re-emerge and must be considered when prescribing or changing a patient’s HAART regimen.