HIV reservoirs are dominated by genetically younger and clonally enriched proviruses
Authors: Kinloch Natalie N. , Shahid Aniqa , Dong Winnie , Kirkby Don , Jones Bradley R. , Beelen Charlotte J. , MacMillan Daniel , Lee Guinevere Q. , Mota Talia M. , Sudderuddin Hanwei , Barad Evan , Harris Marianne , Brumme Chanson J. , Jones R. Brad , Brockman Mark A. , Joy Jeffrey B. , Brumme Zabrina L. 
Published: April 2023

In order to cure HIV, we need to better understand the within-host evolutionary origins of the small reservoir of genome-intact proviruses that persists within infected cells during antiretroviral therapy (ART). Most prior studies on reservoir evolutionary dynamics however did not discriminate genome-intact proviruses from the vast background of defective ones. We reconstructed within-host pre-ART HIV evolutionary histories in six individuals and leveraged this information to infer the ages of intact and defective proviruses sampled after an average >9 years on ART, along with the ages of rebound and low-level/isolated viremia occurring during this time. We observed that the longest-lived proviruses persisting on ART were exclusively defective, usually due to large deletions. In contrast, intact proviruses and rebound HIV exclusively dated to the years immediately preceding ART. These observations are consistent with genome-intact proviruses having shorter lifespans, likely due to the cumulative risk of elimination following viral reactivation and protein production. Consistent with this, intact proviruses (and those with packaging signal defects) were three times more likely to be genetically identical compared to other proviral types, highlighting clonal expansion as particularly important in ensuring their survival. By contrast, low-level/isolated viremia sequences were genetically heterogeneous and sometimes ancestral, where viremia may have originated from defective proviruses. Results reveal that the HIV reservoir is dominated by clonally-enriched and genetically younger sequences that date to the untreated infection period when viral populations had been under within-host selection pressures for the longest duration. Knowledge of these qualities may help focus strategies for reservoir elimination.

Importance

Characterizing the HIV reservoir that endures despite antiretroviral therapy (ART) is critical to cure efforts. Our observation that the oldest proviruses persisting during ART were exclusively defective, while intact proviruses (and rebound HIV) all dated to the years immediately pre- ART, explains why prior studies that sampled sub-genomic proviruses on-ART (which are largely defective) routinely found sequences dating to early infection, whereas those that sampled viral outgrowth sequences found essentially none. Together with our findings that intact proviruses were also more likely to be clonal, and that on-ART low-level/isolated viremia originated from proviruses of varying ages (including possibly defective ones), our observations indicate that: 1) on-ART and rebound viremia can have distinct within-host origins, 2) intact proviruses have shorter lifespans than grossly-defective ones, and therefore depend on clonal expansion for persistence, and 3) the HIV reservoir, being overall genetically younger, will be substantially adapted to within-host pressures, complicating immune-based cure strategies.