HIV-1 persistence: molecular characterization in blood and tissues under antiretroviral therapy

The human immunodeficiency virus (HIV-1)’s DNA integrates into the cellular genome and establishes a reservoir that persists despite efficient antiretroviral therapy (ART). This reservoir constitutes the main obstacle for viral eradication.

It has been previously reported that early ART allows a more important decay of total HIV-1 DNA compared with ART initiated during chronic infection, however, its impact on the dynamics of integrated HIV DNA, the most stable form, is not well explored. In this light, we conducted a retrospective study aiming to describe the kinetics of the decay of integrated HIV DNA depending on the timing of ART initiation. We included 41 participants, diagnosed during primary infection and treated during the chronic phase (ARNS PRIMO cohort) and 92 participants diagnosed and treated during primary infection (ANRS Optiprim-2 trial). We quantified both total and integrated HIV DNA in blood samples taken at primary infection, treatment initiation and one to two years under ART. We showed that early ART limits the stock of integrated HIV DNA in immune cells, thus validating a previously proposed model by our team (Trémeaux et al., 2019). However, deferred ART had little impact on this form. Our study highlights the importance of early ART as a criterion for selecting good candidates for future remission strategies (Mchantaf et al., 2023).

Persistent HIV DNA could harbor drug resistance mutations (DRMs) to ART. However, cases of antiretroviral drugs recycling have been reported in certain studies, despite some DRMs being described in circulating forms during past therapeutic failures. In the second part of this work, I was interested in studying the persistence of these DRMs in the reservoir and the intactness of proviruses harboring these mutations. We studied retrospectively, the case of i) six subjects virologically controlled under Cabotegravir/Rilpivirne (RPV) with a history of therapeutic failure to an agent of the antiretroviral classes and ii) one who experienced virological failure under this combination. We characterized archived proviruses in blood samples collected before the initiation of CAB/RPV with near-full length genome sequencing in limiting dilution and we analyzed the intactness of proviruses harboring DRMs. We also sequenced RT and IN genes with amplicon approach. DRMs to CAB/RPV were found archived in the blood of 4 participants under virological control. The majority of these DRMs were harbored by proviruses in minority (<20%) and defective. Only H221Y, associated to resistance to RPV was found on intact genomes at a low mutational load. Our results highlight the importance of evaluating the intactness of proviruses in interpreting resistance genotyping on DNA (Mchantaf et al., 2024).

HIV reservoirs are mainly present in deep tissues. In the third part, I was interested in studying lymph nodes, PBMCs, adipose tissues, sperm and rectum in 20 participants successfully treated with dolutegravir (ANRS DOLUVOIR study). We studied the levels of HIV DNA, HIV transcripts and the eventual presence of DRMs in each anatomical compartment. The levels of infection and transcription were the highest in lymph nodes. Some DRMs were found in tissues, however they were either harbored by defective genomes (hypermutated) or not associated with a major resistance to dolutegravir. Our results show that the persistence of viral transcription in deep tissues is not associated with selection of DRMs.

The persistence of HIV reservoir depends on the timing of ART initiation. The study of viral reservoirs is complex as it needs to take into consideration its dynamic character, anatomical localization (tissue/blood) and intactness/defectiveness of proviruses. The impact of DRMs on treatment response should be revisited in the light that most proviruses are defective.

Keywords: HIV-1 reservoir,  integrated HIV-1 DNA,  early treatment,  antiretroviral drugs resistance,  HIV-1 DNA genotyping,  full-length genome sequencing,  viral persistence,  defective genome,  dolutegravir,  tissue reservoirs