Background:
Latent HIV-1 persists in a population of resting CD4+ T cells (rCD4s) harboring a transcriptionally silent provirus. It was previously shown that this latent reservoir decays slowly in individuals on antiretroviral therapy (ART) over the first seven years of therapy with a half-life of 44 months (Siliciano, Nat Med, 2003). However, it is unclear if the latent reservoir continues to decay in people on very long-term ART.
Methods:
We recruited 11 people living with HIV (PLWH) who had been on suppressive ART for at least 20 years. To determine reservoir size and the frequency of inducible, replication-competent HIV-1, we performed quantitative viral outgrowth assays (QVOAs) with purified rCD4s. Wells scored positive for viral outgrowth were subjected to RNA extraction and full length env sequencing to explore the clonality of the reservoir. Additionally, we extracted genomic DNA from rCD4s to quantitate the proportion of intact and defective HIV proviruses using the intact proviral DNA assay (IPDA).
Results:
Viral outgrowth was detected in all study participants in the QVOA. The frequency of rCD4s harboring inducible, replication competent HIV ranged from 0.03-16.2 infectious units per million (IUPM) and was highly correlated with the number of intact proviruses detected in the IPDA. Intact proviruses detected by the IPDA ranged from 2.8-168.1 copies per million cells and comprised < 10% of the total number of proviruses detected using this assay. Full-length env sequencing results suggest a trend in several individuals towards a high proportion of identical sequences.
Conclusions:
In this study, we demonstrated that the latent reservoir in PLWH on ART for more than 20 years harbors inducible, replication-competent provirus. The range of IUPM values measured in this study are similar to those measured previously, indicating that over a very long time interval, the reservoir does not decay significantly. This is likely because decay processes are counterbalanced by infected cell proliferation. Full-length env sequences exhibited limited diversity in some individuals, implying that expanded T cell clones harboring identical proviruses contribute to long-term reservoir persistence. Ultimately, these data provide evidence that continued ART is necessary even following 20+ years of ART.