CROI 2024 Abstract eBook

Abstract eBook

Poster Abstracts

hypothesized that completed, multiply spliced, and intact HIV RNA will decay at a faster rate than initiated, 5'elongated, or defective HIV RNA. Methods: CD4+T cells were isolated from blood before ART (T1) and 6 mo (T2) ± 1 year after suppressive ART from 9 PWH (Treat Acute Cohort). U3-U5, TAR, R-U5/Gag and Pol HIV DNA regions, as well as 5'defective (Psi−RRE+), 3'defective (Psi+RRE-), and intact (Psi+RRE+) proviruses, were measured by ddPCR. HIV transcripts, including total initiated (TAR), 5'elongated (R-U5/Gag), mid-transcribed (Pol, unspliced), completed (U3-polyadenylated), multiply spliced (Tat-Rev), 5'defective, 3'defective, and intact HIV RNA, were measured by RT-ddPCR. We also calculated ratios of each HIV RNA to the corresponding HIV DNA (to account for proviral mutations) and ratios of one HIV RNA to another (to measure blocks to transcription). Results: In untreated infection, we observed an excess of initiated over 5'elongated HIV RNA (median 5'elongated/initiated=0.19; P=0.03) but no significant difference between levels of 5'elongated and completed HIV transcripts. ART induced progressive reductions in initiated (median T1/ T2=11; P=0.06), 5'elongated (84; P=0.03), mid-transcribed (143; P=0.03) and completed (503; P=0.03) HIV transcripts. These trends persisted after normalization to the corresponding HIV DNA. Completed transcripts decayed faster than initiated (P=0.03) or 5'elongated transcripts (P=0.03). The ratio of completed/5'elongated HIV RNA was lower at T2 than T1 (P=0.03). ART also reduced intact, 3'defective, and 5'defective HIV RNA (P<0.05 for all). Intact transcripts tended to decay faster than Psi+RRE- HIV RNA (P=0.06). After normalizing to the corresponding HIV DNA, ART reduced Psi+RRE- HIV RNA/DNA (median T1/T2=11.3; P=0.03) and tended to reduce intact HIV RNA/DNA (376.9; P=0.09). Conclusion: Although ART does not target HIV transcription, the pattern of HIV transcriptional processivity differed between untreated infection (less of a block to completion) and early ART suppression. ART reduced completed HIV RNA more than initiated or 5'elongated HIV RNA, and tended to reduce intact HIV RNA more than 3'defective HIV RNA. These findings suggest distinct clearance of cells depending upon HIV RNA processivity and absence of proviral mutations. Multiomics of Detectable vs Undetectable Monocyte Cell-Associated HIV RNA During Acute HIV Michael J Corley 1 , Ivo Sahbandar 1 , Phillip Chan 2 , Alina P. Pang 1 , Nittaya Phanuphak 3 , Carlo P. Sacdalan 3 , Sandhya Vasan 4 , Lydie Trautmann 4 , Serena S. Spudich 2 , Lishomwa Ndhlovu 1 , for the SEARCH010/RV254 Study Group 1 Weill Cornell Medicine, New York, NY, USA, 2 Yale University, New Haven, CT, USA, 3 SEARCH, Bangkok, Thailand, 4 Henry M Jackson Foundation, Bethesda, MD, USA Background: Monocytes play a significant role in the early immune response during acute HIV infection (AHI), and the extent myeloid cell dysregulation has implications for long-term central nervous system (CNS) outcomes. We hypothesized that monocytes carrying HIV RNA would show increased transcriptional dysregulation during AHI. Methods: We isolated ultra-high purity monocytes from 25-40 million PBMC aliquots of 17 participants in the Thai RV254/SEARCH010 AHI cohort obtained prior to ART during AHI (Fiebig I-V) to measure genome-wide transcriptome expression and epigenetic profiles and assess the detection of cell-associated (CA-) HIV RNA. Mann Whitney and T tests examined demographic differences between those with detectable and undetectable monocyte CA-HIV RNA. Differential expression analyses compared participants with detectable versus undetectable HIV RNA using an FDR adjusted P value. Results: Participants had a median age of 26 yrs, median log 10 plasma viral load of 5.55 copies/mL (IQR: 4.87-6.51), CD4+ T cell count of 451 cells/mm 3 (324.5-644), and CD4/CD8 ratio of 0.64 (0.37-1.09). 11/17 participants had detectable CSF HIV RNA levels in supernatant. 41% were in Fiebig stages I/II, and 59% in stages III-V. 8/17 (47%) had detectable monocyte HIV CA-RNA. Those with detectable monocyte HIV RNA trended higher in plasma viral load (6.17 vs 4.79; p=0.05) and lower in CD4/CD8 ratio (0.52 vs 1.02; p=0.06), but there was no difference in CD4 count or Fiebig stage between groups. 70 genes were significantly differentially expressed in monocytes comparing participants with detectable versus undetectable monocyte HIV RNA at an FDR adjusted P < 0.05. Notably, expression of immunomodulatory inflammatory gene CD38, cell surface receptor gene CD40, interferon-induced gene MX1, and viral RNA sensor IFIH1 were significantly higher in participants with detectable versus undetectable monocyte cell-associated HIV RNA. Moreover, we identified that expression of pro-apoptotic BCL-2 protein family gene PMAIP1 and transcription

perforin, and granulysin. Shared clonotypes of HIV RNA+ cells (expressing the same TCR) were found in multiple tissue compartments; these cells showed distinct surface phenotypes and transcriptional profiles characteristic of their tissue site of residence. Conclusion: Our study presents new tools that improve detection of HIV RNA+ cells at the single-cell level, enabling us to establish an atlas of HIV-transcribing cells from PWH on suppressive ART. HIV-transcribing cells clonally expand and disseminate to multiple tissue sites where they adopt tissue site-specific features. HIV-1 Transcriptional Activity Is Largely Driven by Defective Proviruses Mareva Delporte 1 , Evy E. Blomme 1 , Evelien De Smet 1 , Maxime Verschoore 1 , Marie-Angélique De Scheerder 2 , Sofie Rutsaert 1 , Sarah Gerlo 1 , Wim Trypsteen 1 , Linos Vandekerckhove 1 1 Ghent University, Ghent, Belgium, 2 Ghent University Hospital, Ghent, Belgium Background: The majority of cells that are latently infected with HIV-1 do not produce viral RNA, making it difficult for the immune system and current antiretroviral therapy (ART) to target them. To study the underlying mechanisms governing latent HIV-1 infection, a panel of reverse transcription droplet digital polymerase chain reaction (RT-ddPCR) assays specific for different HIV-1 transcripts has been described that define distinct blocks to transcription. Methods: We designed the Rainbow transcriptional HIV-1 RNA 4-plex digital PCR assay, compatible with the QIAcuity 5-plex system, to quantify elongated (long LTR), unspliced (pol), multiple-spliced (Tat-Rev) and completed transcripts (polyA). To evaluate technical performance, a 2-fold dilution curve was prepared containing J-Lat copy DNA (cDNA), ranging from 0.005 to 2.5 ng input, and measured in 20 replicates. This assay was then applied to samples from 40 people living with HIV (PLWH) on ART. Additionally, the HIV-1 DNA reservoir was quantified by the Rainbow proviral HIV-1 DNA assay. Intactness levels were defined by two target regions: psi and env (IPDA), and by four target regions: psi, env, gag and pol (D4PCR). Levels of defective proviruses were measured by the difference between total and intact HIV-1 DNA levels (D4PCR). Results: Linear quantification was observed for each HIV transcript (slope: 0.98). The Limit of Detection (LoD) was <10 copies/well for all HIV-1 RNA transcripts, except poly A (average: 11.1 copies/well, range: 5.6-24). The Limit of Blank (LoB) was calculated by using 90 negative template control (NTC) samples, resulting in an LoB of 1 copy/well or less in long LTR, pol and Tat-Rev. The LoB of polyA was 15.75 copies/well. In samples from 40 PLWH, HIV-1 RNA levels were compared to total and intact HIV-1 DNA levels (Figure 1). We found that intactness levels by IPDA correlated better with HIV-1 RNA transcripts than intactness levels by D4PCR. Interestingly, defective proviruses showed a stronger correlation with HIV-1 RNA transcripts compared to intact proviruses. Conclusion: The Rainbow proviral HIV-1 DNA and transcriptional HIV-1 RNA assay allow us to map the characteristics of the viral reservoir by using a minimal amount of sample. This analysis resulted in a strong correlation between defective proviruses and HIV-1 RNA transcripts, suggesting that the HIV transcriptional activity is largely driven by defective proviruses.

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Dynamics of Different Proviruses and HIV Transcripts After Acute ART Treatment Julie Janssens 1 , Sun Jin Kim 1 , Adam Wedrychowski 1 , Gayatri N. Kadiyala 1 , Satish K. Pillai 2 , Timothy J. Henrich 2 , Nadia R. Roan 3 , Steven G. Deeks 2 , Sulggi A. Lee 2 , Steven A. Yukl 2 1 San Francisco VA Medical Center, San Francisco, CA, USA, 2 University of California San Francisco, San Francisco, CA, USA, 3 Gladstone Institute of Virology and Immunology, San Francisco, CA, USA Background: Different subsets of HIV-transcribing cells may contribute to immune activation and rebound. We investigated the dynamics of proviruses and different HIV transcripts after initiation of ART during acute infection. We

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