In-depth assessment of the HIV-1 viral reservoir in early treated individuals

Background: The integration site and intactness of proviral genomes remain poorly characterized in early treated individuals on antiretroviral therapy (ART). Moreover, the inducibility of the viral reservoir in these individuals is hampered by the lack of latency reversing agents (LRA) capable of inducing potent HIV reactivation. Here, we did an in-depth assessment of the total and inducible viral reservoir in early treated individuals. Methods: We collected leukaphereses from 8 individuals treated during acute infection (Fiebig II-III: n=6; Fiebig IV: n=1; Fiebig V: n=1) who received ART for a median of 0.96 years (0.49-1.93 years). Total HIV DNA and intact HIV proviral DNA (IPDA) were assessed by multiplex digital PCR. Near full-length (NFL) proviral sequences and integration sites were obtained by matched integration site and proviral sequencing (MIP-Seq). Following a 24h-stimulation with a Tat mimetic (Tat#1) and PMA, the frequency of p24-expressing cells and their phenotype were assessed by HIV-Flow. Results: In total, 252 integration sites and 64 proviral genomes were obtained. Clonally expanded cells were retrieved in only 4 out of 8 participants and accounted for 5% of total integration sites. NFL genome analyses revealed that 9% of the proviruses were intact (91% defects: 2% inversions, 53% large internal deletions, 17% hypermutations, 9% PSI/MSD defects, 9% premature stopcodon/frameshift). The analysis of a 4.5 kb region at the 3’ end of the provirus (HXB2 positions 5089-9602, n=59) revealed that the intra-individual genetic diversity is limited (average genetic distance=1.9 bp). Following PMA/Tat#1 stimulation, the frequency of p24+ cells ranged between 0.4-20 per million CD4 T cells. p24-expressing cells were enriched in effector memory T cells (p=0.06). Interestingly, a significantly higher fraction of p24+ cells displayed a naïve phenotype in early treated individuals compared to chronically treated individuals (p=0.03). Conclusions: Collectively, these data indicate that the contribution of clonal expansion to the persistence of the viral reservoir in early treated individuals is minimal after 1 year of treatment. We report a combination of LRAs that allows for the successful detection of the inducible reservoir in early treated individuals on ART. Single-cell assessment of the viral composition of the inducible reservoir should provide further insights into the persistence of those cells during ART

Potent latency reversal by Tat RNA-containing nanoparticle enables multi-omic analysis of the HIV-1 reservoir

Abstract The development of latency reversing agents that potently reactivate HIV without inducing global T cell activation would benefit the field of HIV reservoir research and could pave the way to a functional cure. Here, we explore the reactivation capacity of a lipid nanoparticle containing Tat mRNA (Tat-LNP) in CD4 T cells from people living with HIV undergoing antiretroviral therapy (ART). When combined with panobinostat, Tat-LNP induces latency reversal in a significantly higher proportion of latently infected cells compared to PMA/ionomycin (≈ 4-fold higher). We demonstrate that Tat-LNP does not alter the transcriptome of CD4 T cells, enabling the characterization of latently infected cells in their near-native state. Upon latency reversal, we identify transcriptomic differences between infected cells carrying an inducible provirus and non-infected cells (e.g. LINC02964, GZMA, CCL5). We confirm the transcriptomic differences at the protein level and provide evidence that the long non-coding RNA LINC02964 plays a role in active HIV infection. Furthermore, p24+ cells exhibit heightened PI3K/Akt signaling, along with downregulation of protein translation, suggesting that HIV-infected cells display distinct signatures facilitating their long-term persistence. Tat-LNP represents a valuable research tool for in vitro reservoir studies as it greatly facilitates the in-depth characterization of HIV reservoir cells’ transcriptome and proteome profiles