CROI 2025 Abstract eBook

Abstract eBook

Poster Abstracts

Results: ARCs exhibited robust expression of CD8-like cytotoxic programs, contrasting with autologous uninfected clones (Figure 1A; 3 donors; ARCs n=3; Uninfected clones n=10). ARCs also displayed higher expression of interferon response genes. >90% of cells within each ARC were confirmed to be GzmA+, TNFa+ Th1s (IFNg+). While most of the cells from ARCs had low to no expression of HIV transcripts, a subpopulation of cells from donor 1 ARC displayed high expression of HIV (Figure 1B). This HIV-hi population had markedly lower expression of cytotoxic and interferon response genes. LRA-treatment of ARCs resulted in limited latency reversal (Donor 2 ARC: 0.05% +/- 0.02, Donor 3 ARC: 2.12n%+/- 2.25 Gag+ cells after treatment). LRA-treated cells that released GzmA from 2 donor ARCs showed 2.2-3 fold lower Gag-expression compared to cells that did not release granzyme. Conclusions: Each ARC was enriched for cytotoxic and interferon response programs and determined to be inflammatory-like Th1s expressing high levels of TNFa and granzyme. A small subset of cells expressing HIV transcripts within 1 ARC differed from the majority of cells by having significantly lower expression of cytotoxic and interferon response genes. LRA-treated cells from 2 other ARCs displayed increased latency reversal in cells that did not undergo degranulation, suggesting differential latency reversal capacity based on cell-intrinsic cytotoxic properties. Our results highlight the value of using ARCs to evaluate the contribution of the immunophenotypes of reservoir-harboring cells to HIV persistence. The Paucity of Clonal SIV Proviral Sequences During Early ART Treatment Margaret Hallmets 1 , Celeste Coleman 1 , Chrysostomos Perdios 1 , Anna T. Brown 1 , Clara Krzykwa 1 , Lori Rowe 1 , Brandon Keele 2 , Charles Goodman 2 , Joseph C. Mudd 3 1 Tulane National Primate Research Center, Covington, LA, USA, 2 Frederick National Laboratory for Cancer Research, Frederick, MD, USA, 3 Tulane University, Metairie, LA, USA Background: The persistent HIV-1 reservoir is comprised of individual proviral clones with a corresponding integration site. The proviral landscape in the blood of long-term ART-suppressed persons with HIV-1(PWH) is often dominated by identical sequences, signifying antigen-driven proliferation. The clonal structure of the SIV reservoir during ART suppression is less well-characterized, particularly in the first 2 years of ART, which is commonly when HIV-1 cure strategies undergo pre-clinical testing. Methods: We employed a linker-mediated PCR-based assay coupled with next generation sequencing to longitudinally assess proviral integration sites in the blood of SIVmac239M-infected macaques (n=3) at 3 and 10 months of ART. Results: We sampled a total of 357 SIV integration sites during both ART time points. Of these, we encountered predominantly single unique proviral sites (Fig. A). 39 (10.9%) proviral clones were found to be identical, with no statistical difference in the number of identical integration sites between the two timepoints (p = 0.9934). We also did not observe clonality increase from 3 to 10 months of ART, a phenomenon known to occur in PWH over long-term ART intervals. We observed a very low frequency of SIV integration sites shared between 3- and 10-month ART timepoints (1.1%) (Fig. B). Shared clones however had a high likelihood to be clonal at 3 or 10 months, confirming cellular proliferation as a driver of reservoir persistence. One animal was found to have 7 clones at 3 months, ~10% of integration sites sampled. This clone decreased to 2 clones (4.9%) at 10 months. This integration site was within the DDX49 gene; an RNA Helicase that has previously been associated with cellular proliferation. Conclusions: Our data indicate an absence of dominating SIV proviral clones up to 10 months of ART. Suggesting the SIV reservoir during periods of pre clinical therapeutic testing is polyclonal, and predominantly maintained by homeostatic rather than antigenic proliferation. At 3 months of ART, one animal displayed a highly expanded clone integrated into DDX49. Given that early ART reservoirs are orders of magnitude larger than those of long-term suppression, this clone is likely to be massive. The frequency of this clone contracted at 10 months, suggesting that large clones within genic regions may be under negative selection pressure during reservoir decay. Overall, the data suggest that the SIV proviral landscape is markedly distinct from that of long-term suppressed PWH. The figure, table, or graphic for this abstract has been removed.

clone carrying a solo-LTR, suggesting that the solo-LTR clone originated from an ancestral cell or cells carrying the provirus with internal sequence that underwent homologous recombination to generate a solo-LTR in a daughter clone. Identifying and exploiting the mechanisms that promote the formation of solo- LTRs could enable inactivation of proviruses with internal sequences, including intact proviruses. Chromosomal Integration Site Profile of HTLV-1-Infected Cells Is Distinct From HIV-1-Infected Cells Alex Hochroth 1 , Nicolás Ducasa 2 , Maxime Bellefroid 1 , Ce Gao 1 , Chloe M. Naasz 1 , Xu G. Yu 1 , Mirna Biglione 2 , Mathias Lichterfeld 1 1 Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA, 2 Instituto de Investigaciones Biomédicas en Retrovirus y SIDA, Buenos Aires, Argentina Background: Both HTLV-1 and HIV-1 cause a lifelong incurable infection, but mechanisms of viral persistence may vary. In HIV-1 infection, prior data demonstrated a progressive accumulation of intact proviruses in heterochromatin during long-term ART, likely as a result of immune-mediated selection that promotes viral persistence in repressive chromatin. Whether and how proviral integration site locations influence persistence of HTLV-1-infected cells remains less clear. Methods: We analyzed DNA from 5 adult people living with HTLV-1, four with long-term asymptomatic infection and one with early-onset Adult T-cell leukemia (ATL). Proviral sequences and corresponding chromosomal locations were identified using adapted protocols of FLIP-Seq and MIP-Seq and compared to data from long-term ART-treated persons with HIV-1 or elite controllers (EC). Results: Average proviral loads were 111,661 copies/million PBMC among asymptomatic carriers, and 749,554 viral copies in the participant with ATL. DNA analysis yielded 132 proviral sequences and 24 integration sites across all 5 participants. Approximately 77% of HTLV-1 proviruses showed no major structural defects and were likely replication-competent. The proportion of clonally expanded intact sequences was lower in HTLV-1 study participants, averaging only 34% of all intact sequences, compared to greater than 80% in persons long-term ART or EC. Observed integration sites in HTLV-1 study participants were mostly genic, representing 63% of all integration sites. Non-genic sites composed 30% of the remaining reservoir, while proviruses integrated in satellite or centromeric regions were only found in 7% of the reservoir cells. No integration sites were found in ZNF-family genes for HTLV-1, in sharp contrast to HIV-1 infection where ZNF genes represent hotspots for persistence of intact proviruses. There was no evidence for preferential persistence of intact HTLV-1 genomes in oncogenes or proliferation-associated genes in asymptomatic carriers and in the ATL case. Conclusions: Integration sites of intact HTLV-1 genomes were markedly more diverse than in persons with HIV-1 on long-term ART or EC, and less likely to accumulate in heterochromatin regions. These data suggest that HTLV-1 infected cells are under more limited selection pressure and/or less dependent on the chromatin microenvironment for long-term persistence. Relative to HTLV-1 infection, HIV-1 reservoir cells seem more vulnerable to host immune pressure. Reservoir-Harboring T-Cell Clones Have a Cytotoxic Immunophenotype That May Affect HIV Inducibility Alberto Herrera 1 , Isabella A. T. M. Ferreira 1 , Tan Thinh Huynh 1 , Emily Stone 1 , Noemi L. Linden 1 , Cintia Bittar 2 , Marina Caskey 2 , Michel Nussenzweig 2 , R. Brad Jones 1 1 Weill Cornell Medicine, New York, NY, USA, 2 The Rockefeller University, New York, NY, USA Background: The HIV reservoir is enriched for clonal CD4+ T-cells harboring intact proviruses after years of antiretroviral therapy (ART). In this study, we characterize the immunophenotypes of ‘authentic reservoir-harboring clones’ (ARCs) isolated and propagated from people living with HIV (PLWH) and aim to understand the contribution of these profiles to persistence. Methods: Individual ARCs from PLWH on ART (n=3) were isolated by limiting dilution of memory CD4+ T-cells and propagated with irradiated feeders and anti-CD3/CD28 stimulation in media containing ARVs and bNAbs. Wells were screened for the presence of ARCs by Gag-p24 ELISA and infected cell purity was validated by IPDA and single-cell TCR-sequencing. Transcriptional, surface protein profiles and HIV-expression of ARCs were characterized by CITEseq and confirmed by flow cytometry. Latency reversal activity and granzyme release from ARCs were assessed using PMA/Ionomycin.

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