CROI 2025 Abstract eBook

Abstract eBook

Poster Abstracts

553

Targeting of HIV-Infected Clones by Cognate Peptide Stimulation and Antiproliferative Drugs Joel Blankson, Joel Sop, Tyler Beckey, Isha Gurumurthy, Kellie Smith, Francesco R. Simonetti, Filippo Dragoni The Johns Hopkins University School of Medicine, Baltimore, MD, USA Background: Clonal expansion of HIV infected CD4+ T cells is a barrier to HIV eradication. Previous studies suggested that blocking cell proliferation can accelerate reservoir decay. We recently described a marked reduction in clonally expanded infected CD4+ T cells in a patient with elite control (ES24) after chemoradiation for lung cancer with paclitaxel and carboplatin. We tested the hypothesis that this phenomenon was due to a higher susceptibility of frequently proliferating CD4+ T cell clones to the chemotherapeutic drugs. Methods: We studied a large CD4+ T cell clone with replication-competent provirus integrated into the ZNF721 gene (ZNF721i), which has been linked to the persistence of intact proviruses. We stimulated CD8-depleted PBMCs from ES24 with the cognate peptide for the ZNF721i clone (peptide 61: Gag 241-255) for 20 hours on day 1. On day 5, the cells were exposed to paclitaxel and/or carboplatin, or the antiproliferative drug, mycophenolate mofetil (MMF). The cells were harvested on days 7 to 9. The frequency of total HIV DNA and the ZNF721i cells was determined by LTR and integration site-specific digital PCR. Single-cell transcriptome analysis and T cell receptor sequencing was also performed on day 7 CD4+ T cells. Results: There was a 25-to-30-fold increase in the frequency of ZNF721i cells after treatment of cells with peptide 61. The expanded ZNF721i cells had the phenotype of proliferating (MKI67, CDT1) cytotoxic (CCL5, GZMK, NGK7) CD4+ T cells. Both paclitaxel and MMF completely abrogated peptide induced expansion of ZNF721 cells, resulting in a transcriptome signature of cell cycle arrest in the G2 phase. In contrast, carboplatin had no effect. Other peptide-reactive clonotypes were similarly affected, but the drugs did not deplete expanded clones that were not specific for peptide 61. Conclusions: This strategy of antigen-specific stimulation followed by treatment with an antiproliferative agent may lead to the selective elimination of clonally expanded HIV infected cells. Sequencing of HIV-1 Proviruses in Large Infected T-Cell Clones Reveals a Predominance of Solo-LTRs Dimiter Demirov 1 , Shahinur Akter 2 , Shadab Parvez 2 , Leah Danielle Brandt 3 , Elias Konstantine Halvas 3 , Sean C. Patro 1 , Jason W. Rausch 2 , John W. Mellors 3 , Xiaolin Wu 4 , Mary Kearney 2 1 Leidos Biomedical Research, Inc, Frederick, MD, USA, 2 National Cancer Institute at Frederick, Frederick, MD, USA, 3 University of Pittsburgh, Pittsburgh, PA, USA, 4 National Institutes of Health, Bethesda, MD, USA Background: Most methods of amplifying and sequencing proviruses do not capture the flanking long terminal repeats (LTRs) because of technical difficulties related to sequence identity between these elements. Consequently, defective HIV-1 proviruses consisting of a single LTR (solo-LTR), a common proviral structure across retroviruses, are not detected and thus their frequency and significance are poorly understood. Here we developed a new method able to detect solo LTRs and characterize their abundance in PBMC samples from PWH on ART. Methods: Several established methods and a new approach were used to sequence full-length proviruses in the largest HIV containing T cell clones in 3 adult PWH with detectable viremia on long-term ART (9-23 years). The methods included integration site analysis (ISA), multiple displacement amplification single genome sequencing (MDA-SGS), integration site qPCR (IS-qPCR), and a new droplet-based PCR method in which HIV-host “junction” primers are used to amplify full-length proviruses by nested PCR. Results: Full-length amplification and sequencing of proviruses in 24 of the largest infected T cell clones in three PWH revealed 2 intact and 22 defective proviruses, of which 7 contained large internal deletions and 12 were solo-LTRs. MDA-SGS and ISA identified a provirus with internal HIV sequence, which included Psi, that had the same integration site as a clone carrying solo-LTR proviruses. This finding suggests that the progenitors of proviruses that undergo recombination to become solo LTRs may persist at low levels on ART. Conclusions: Our results reveal that solo-LTRs are the most common proviral structure found in large HIV-infected cell clones in three PWH on ART. Using droplet-based PCR with HIV-host junction primers, several solo-LTRs were characterized that would have otherwise been undetectable. One provirus with internal sequence was found to share an integration site with a large

and immunophenotypic profiles were assessed by CITE-Seq, while antigen specificities were queried by ELISPOT. Results: Each of the four ARCs contained a single intact provirus integrated into either ZNF561 , ZNF486 , ZNF721 , or ATPB4 . The ZNF721 ARC was poorly proliferative, exhibiting minimal reactivation (0.2±0.1% Gag+) in response to anti-CD3/anti-CD28 stimulation. The ZNF486 ARC was deeply latent, with Gag expression of 0.04±0.3% upon reactivation with anti-CD3/CD28 (0% at baseline). The ZNF721 ARC, however, showed elevated basal Gag expression (1.16±1.18%) and modest reactivation with bryostatin-1 (3.27±1.22%). Distinct transcriptional and phenotypic profiles were revealed by CITE-Seq analysis, but shared features emerged: cytotoxicity and the expression of interferon stimulated genes. CTL killing assays using peptide-pulsed targets demonstrated differential susceptibilities across the ARCs: the ATPB4 ARC resisted killing, ZNF486 was highly susceptible, and ZNF561 and ZNF721 showed moderate susceptibilities. Notably, the ZNF721 ARC had a proliferative advantage over autologous uninfected CD4+ T cells (Figure), was determined to be EBV specific, and killed EBV-transformed B cells. Conclusions: Each ARC featured distinct mechanisms of persistence. The ATPB4 ARC was resistant to CTL-mediated killing, poorly proliferative, but displayed the highest HIV expression. In contrast, ZNF486 was deeply latent, while ZNF721 was highly proliferative. ZNF561 was intermediate for each of these properties. ARCs serve as valuable models for understanding HIV persistence mechanisms and may guide target interventions. The figure, table, or graphic for this abstract has been removed. Latent HIV Clonal Dynamics: Integration Sites and Their Role in Maintenance of Latency In Vivo Virender K. Pal 1 , Frauke Mücksch 2 , Ali Danesh 3 , Marie Canis 1 , Tan Thinh Huynh 3 , Thomas R. Dilling 3 , Itzayana G. Miller 3 , Theodora Hatziioannou 1 , R. Brad Jones 3 , Guinevere Lee 3 , Paul Bieniasz 1 1 The Rockefeller University, New York, NY, USA, 2 Heidelberg University, Heidelberg, Germany, 3 Weill Cornell Medicine, New York, NY, USA Background: There is a paucity of understanding of how HIV latent reservoirs survive and expand, escape immune clearance, and reactivate upon ART cessation. The integration site (IS) of proviral DNA into the host genome could (i) determine the transcriptional status of the provirus and (ii) influence the expansion of infected cells. In this study, we determined the proviral IS associated with clonal expansion and latency of HIV infected cells in an in vivo model. Methods: Memory CD4 + T cells isolated from healthy human donors were infected with dual reporter- tagged, replication-defective HIV-1 to generate large populations (millions) of infected cells, each carrying a single transcriptionally active reporter provirus at a distinct IS. Clonal expansion and proviral reporter expression dynamics of infected cells was analyzed post engraftment in NSG mice. HIV-1 IS were determined by PCR-based amplification of host-viral junction and next-generation sequencing. Results: Following engraftment of HIV-1 infected CD4 + T cells in mice, the fraction of cells in which the HIV-1 reporter provirus was transcriptionally active decreased over time. HIV-1 IS analysis in long-term persistently infected cell clones led to the identification of 646 unique IS and the majority of IS were found in genic regions (82%) of human chromosomes. Comparison of genic IS in multiple animals led to the identification of recurrent integration in genes including NPLOC4, PACS1, NFATC3, NOSIP, and ATF7IP. Gene length and expression level correlated with increased frequency of integration, both in pre-engraftment cells and in expanded cell clones after engraftment. Sorting of cells into populations with active and latent proviruses at late time points after engraftment revealed that IS harboring transcriptionally latent proviruses were more likely to be found in non-genic, pericentric regions. To study and characterize cells with latent HIV-1 proviruses, we successfully generated and expanded single cell clones from engrafted cells, each carrying distinct proviral IS. Here, we observed that proviral reporter expression in latently infected clones was independent of cellular activation status and maintained during in vitro expansion. Conclusions: HIV IS affects proviral transcriptional status, and latent proviruses were found more frequently integrated in non-genic regions. Current and future work involves studying the mechanisms underpinning individual IS-driven clonal survival of infected cells and proviral transcriptional status.

Poster Abstracts

552

554

CROI 2025 145