CROI 2025 Abstract eBook

Abstract eBook

Poster Abstracts

unclear what happens after starting ART in chronic infection or after longer durations of ART. We hypothesized that completed and intact HIV RNA would decay faster than incomplete or defective HIV RNA but would reach a plateau. Methods: CD4+ T cells were isolated from longitudinal blood samples from 10 PWH during untreated chronic infection (T1) and 4 timepoints on suppressive ART (median years after ART: T2=2.2; T3=4.5; T4=6.9; T5=10.5). HIV transcripts, including initiated (TAR), 5’elongated (R-U5/Gag), mid-transcribed (Pol), completed (U3-polyadenylated), multiply spliced (Tat-Rev), 5’defective (Psi RRE+), 3’defective (Psi+RRE-), and intact (Psi+RRE+) RNA, were measured at each time by RT-ddPCR. Results: Before ART, we observed an excess of initiated over 5’elongated HIV RNA (median elongated/initiated=0.16; P=0.01) and of 5’elongated over completed HIV RNA (median completed/elongated=0.03; P=0.002) but no difference between completed and multiply spliced HIV RNA. These differences persisted after normalization to HIV DNA. From T1 to T2, we observed reductions in 5’elongated (median T1/T2=6; P=0.01), mid-transcribed (2; P=0.03), completed (6; P=0.02), and multiply spliced (43, P=0.049) but not initiated HIV RNA. The ratio of elongated/initiated HIV RNA was lower at T2 than T1 (P=0.01). After normalization to HIV DNA, ART reduced completed (median T1/ T2=10; P=0.04) but not elongated or mid-transcribed HIV RNA/DNA. ART also reduced intact and 3’defective HIV RNA (median T1/T2=7.6 and 4.7; P=0.03 for both). From T2 to T3, multiply spliced HIV RNA declined further (P=0.02), and completed HIV RNA tended to decrease (P=0.08). After T3, no further changes were observed. Conclusions: In untreated chronic HIV infection, we observed differences in levels of various HIV RNA regions that may result from blocks to transcription and/or immune selection. Most HIV transcripts declined during the first few years of ART (T1-T2), but with additional years on ART, only multiply spliced and possibly completed HIV RNA continued to decay from T2 to T3. Completed and multiply spliced HIV RNA may be selectively recognized by the immune system, leading to distinct clearance. Low levels of these mature HIV transcripts during long-term ART may present a challenge for latency reversal, while early HIV transcripts persist and might serve as targets for new therapies. Nonsuppressible HIV Viremia May Harbor Replication-Incompetent Viruses With 5' Leader Deletions Zixin Hu 1 , Eva Soledad Piernas 1 , Behzad Etemad 1 , Andy Du 1 , Hieu Nguyen 1 , Abbas Mohammadi 2 , Trevor Tamura 1 , Nikolaus Jilg 3 , Daniel Worrall 1 , Peter Anderson 4 , Jose Castillo-Mancilla 5 , Steven G. Deeks 6 , Athe Tsibris 1 , Daniel Kuritzkes 1 , Jonathan Li 1 1 Brigham and Women's Hospital, Boston, MA, USA, 2 The Valley Health System, Las Vegas, NV, USA, 3 Massachusetts General Hospital, Boston, MA, USA, 4 University of Colorado Anschutz Medical Campus, Aurora, CO, USA, 5 ViiV Healthcare, Brentford, UK, 6 University of California San Francisco, San Francisco, CA, USA Background: Individuals with nonsuppressible viremia (NSV) have persistent low-level viremia despite ART adherence and lack of drug resistance. A subset was reported to harbor deletions in the 5' end of the HIV-1 genome, specifically within the 5' PSI packaging element. We have previously reported three individuals with NSV and plasma variants harboring deletions in the 5’ leader region within otherwise genome-intact viruses. However, it is unclear whether these 5’ leader deletions lead to replication-defective virus. To investigate whether 5'Leader deletions affect HIV replication capacity fitness, we engineered a vector with a 5'Leader deletion and a unique enzyme site (pHIV Δ5'Leader/PmeI) to study viral replication and infectivity. Methods: We created an HIV-1 vector with 5' Leader deletions and introduced a Pme I site with NL4-3 as the backbone. 5'leader amplicons from three NSV participants harboring 5’ deletions were either PCR-amplified or synthesized. Recombinant viruses carrying patient-derived or synthesized 5'Leader deletions of varying sizes were generated via co-transfection in 293T cells. Infectivity was measured using β-galactosidase assays in TZM-bl cells, while relative fitness was evaluated through kinetic assays in MT-2 cells. The sequence integrity of the 5'Leader was confirmed using NGS sequencing of culture supernatants. Results: Recombinant HIV-1 viruses carrying the 5'Leader deletions from three NSV participants harboring 22, 15 and 41 bp deletions (LV4, 8, and 9, respectively) were replication-incompetent. Among viruses carrying synthesized 5'Leader amplicons, all variants containing the LV4, LV8 and LV9 deletions were replication-incompetent. In addition, using viruses harboring 5’ Leader deletions of varying sizes, 5’ Leader deletions as small as 6 nucleotides rendered the virus replication-defective, as long as that deletion encompassed

a portion of the major splice donor site (MSD). The replication activity of the viruses from these participants could be rescued by the introduction of an intact 5’ Leader sequence. Conclusions: Deletions in the 5'Leader region, especially within the major splice donor site, significantly impaired viral replication capacity, these results demonstrate that persistent low-level viremia may be comprised largely of replication-defective virions, with minimal transmission risk. HIV-1 Latency Reversal Augments Persistent HIV-1 Transcription During ART Rowan Hassman, Rachel Raymond, Sidra Wohlwend, Tyler Lilie, Hannah Jordan, Elise Courtney, Athe Tsibris Brigham and Women's Hospital, Boston, MA, USA Background: Antiretroviral therapy (ART) reduces but does not eliminate intracellular HIV-1 transcription in people with HIV (PWH). While proximal virus transcripts can be detected in blood and tissue, the presence of unspliced distal transcripts is less well characterized. Qualitatively, the sequences of HIV-1 caRNA that are induced by latency reversal agents (LRA) are less well studied than their quantitative levels. Methods: To understand if similar parts of the HIV-1 proviral landscape are transcribed during LRA exposure, we used cryopreserved PBMC from three PWH on ART to amplify near-full length (NFL) and env -specific HIV-1 proviral DNA and caRNA single genome amplicons (SGA) before and after a 24-hour exposure to PMA-ionomycin or the LRA boosting combination of low-dose bryostatin (1nM) and romidepsin 20nM. Amplicons were sequenced, assessed for hypermutation, trimmed, and aligned. Neighbor-joining trees with the Tamura-Nei model using HXB2 as an outgroup, 1,000 bootstrapped replicates, and a support threshold of 70% were generated. A convenience sample size of 20 SGAs each for HIV-1 NFL DNA, env-specific DNA, and env caRNA for each condition was used. Results: An average of 100 SGA were generated per participant. HIV-1 DNA contamination was not detected in caRNA. HIV-1 env -containing caRNA were amplifiable from all three participants’ PBMC before LRA exposure; based on our primer locations, these SGA represent unspliced HIV-1 caRNA. These unspliced caRNAs were spread across the phylogenies and included hypermutated sequences. The choice of primers, NFL vs env-specific, affected the detection of hypermutated proviral DNA. We found that LRA boosting and PMAi can activate HIV-1 transcription from similar parts of the virus reservoir. LRA boosting stimulated hypermutated HIV-1 caRNA transcription in some participants, whereas PMAi did not. We found that LRA boosting and PMAi can induce identical HIV-1 mRNA transcripts as occurs when ART is interrupted. Conclusions: Unspliced HIV-1 caRNA of 8-9kb in length, some of which were hypermutated, were detected during ART and were most likely from >1 transcriptionally active cellular clones. HIV-1 latency during treated infection is best described a period of inefficient, but not absent, virus mRNA production in blood. Hypermutants may contribute to HIV-1 caRNA quantifications. Treatment with HIV-1 LRA can induce virus RNA transcription identical in sequence to that produced during treatment interruptions.

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MLN4924 Reduces HIV Transcription, Cell Survival and HIV Infectivity After Latency Reversal In Vitro Cristina C. Vaca, Hannah E. Hudson, Isabelle Clerc, Chisu Song, Richard T. D'Aquila Northwestern University, Chicago, IL, USA Background: Neddylation is essential for post-translational regulation of many cellular processes, including transcription and protein degradation. The NEDD8-activating enzyme inhibitor MLN4924 (MLN) can block degradation of

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