Persistent low-level viremia (LLV) is not uncommon among patients with HIV despite receiving continuous antiretroviral therapy (ART), but the mechanism behind this finding remains unclear. We describe one individual with persistent low-level viremia (200-700 copies/ml) across 16 viral load measurements over >3 years despite ART intensification to a DTG, DRV/r, TAF/FTC ART regimen. We hypothesized that the persistent LLV arose either from an expanded clone of transcriptionally-active reservoir cells or from ongoing viral replication.
Commercial ARV drug levels and resistance genotyping were performed at multiple time points. We performed plasma single-genome sequencing for the Pro-RT region at 3 different timepoints, each 1 year apart. Confirmatory near-full length plasma sequences were obtained at the first time point. We also performed a novel next-generation single-genome proviral sequencing (NG-SGS) assay from PBMCs that combines near-full length proviral amplification and integration site analysis.
The LLV persisted despite detectable plasma ARV levels and the presence of at least 2 fully active ARVs by resistance genotyping. Across all 3 timepoints, 86% of all single-genome plasma sequences were comprised of one viral clone (range 67% – 100% at each time point). Intact near-full length proviruses exactly matching the majority plasma clone were identified, which constituted only 6% of all intact proviruses. Near-full length plasma HIV sequences confirmed the clonality of this population and the lack of known drug resistance mutations. Integration site analysis showed that this provirus is integrated into CD200R1, a gene encoding a transmembrane receptor expressed by CD4+ cells. Interestingly, the majority of intact proviruses consisted of one clonal proviral sequence, constituting 54% of all intact proviruses but only 9% of plasma variants. This intact provirus is integrated into the STAG2 gene, which has critical roles in regulating the chromosome structure and cell division. No evidence of viral evolution or emergence of new drug resistance mutations were detected in plasma over time.
Persistent LLV can arise from the integration of HIV into a transcriptionally-active region of a clonally-expanded CD4+ population without evidence of ongoing viral replication. In this setting, further intensification of the ART regimen is unlikely to be effective and suppression of the LLV will require targeting of this transcriptionally-active reservoir.