CROI 2025 Abstract eBook

Abstract eBook

Poster Abstracts

at the integration site. Little is known regarding solo HIV LTR formation and understanding mechanisms driving their formation will inform efforts to eradicate expression of HIV proteins. Methods: Cell lines containing HIV proviruses (ACH-2, 8E5) and uninfected control (CEM) were maintained in vitro and sub-cloned. HIV DNA and RNA were quantified using multiplexed droplet digital PCR. Proviruses were recovered from cell lines using enrichment through biotin-labeled probes targeting HIV sequence and characterized by long-read sequencing. To screen host proteins required for the solo-LTR formation, we used a yeast genetic system that introduced a suicide gene in a LTR-retrotransposon. The LTR-LTR recombination leads to the knockout of the suicide gene, which allows us to investigate the frequency of LTR-LTR recombination. Results: Using an enrichment and long read sequencing technique, we captured and characterized HIV-containing DNA fragments in latently HIV-1 infected cell lines. In 8E5 cells, we identified a full-length HIV provirus and a solo-LTR with the exact same integration site, demonstrating formation solo-LTR in vitro. PCR analyses with provirus-flanking primers revealed that the 8E5 cell line is a mixture of cells with either intact or solo-LTR proviruses. Analyses of multiple single cell clones containing full-length or solo-LTR HIV, including growth competition assays, revealed cells with solo-LTR exhibit growth advantage over cells containing full-length provirus. With yeast genetic assay we have revealed that the frequency of solo-LTR formation is approximately 10 events per million cells, which is consistent with the findings in the human endogenous retroviruses. Knockout of homologous recombination (HR) genes rad50 and rad52 , orthologs of Human RAD50 and RAD52 , substantially downregulated the frequency of LTR-LTR recombination. Conclusions: Our group have demonstrated that solo-LTR HIV formation occurs in vitro. New cell lines generated in this study are now available to elucidate mechanisms to excise LTR coding sequences. With yeast system, we have identified HR plays crucial role in the formation of solo-LTR. Development and Delivery of Next-Generation Epigenome Editors for Long-Term HIV Silencing Francisco J. Zapatero Belinchon 1 , Valentina Pedrero-Classen 1 , Martin V. Hamann 2 , Zichong Li 1 , Katelyn Luo 1 , Ulrike V. Lange 2 , Melanie Ott 3 1 Gladstone Institute of Virology and Immunology, San Francisco, CA, USA, 2 Leibniz Institute of Virology (LIV), Hamburg, Germany, 3 Gladstone Institutes, San Francisco, CA, USA Background: Although antiretroviral therapy (ART) has extended the lifespan of people living with HIV (PLWH), it cannot eliminate genome-integrated HIV reservoirs. Approximately 81% of the reservoir remains transcriptionally active, driving immune activation and rapid viral rebound. Silencing these transcriptionally active reservoirs is crucial. Recent studies highlight that DNA and histone methylation can effectively silence cellular transcription, keeping retroviruses in a “deep-latent” state. Leveraging these insights, we developed BrecOFF, a novel HIV-specific epigenome editor, to achieve durable silencing of transcriptionally active HIV. Methods: BrecOFF is a 148 kDa HIV-specific epigenome editor that integrates the de novo DNA methylation machinery DNTM3A-3L with the KRAB transcriptional repressor domain KOX1. To improve targeting specificity and reduce size, the ~160 kDa dCas9 component of CRISPRoff was replaced with a ~38 kDa catalytically inactive version of Brec1 (dBrec1), an HIV-specific recombinase targeting a conserved 34-bp region in the HIV long terminal repeat (LTR). We evaluated BrecOFF’s ability to bind the HIV LTR and inhibit transcription both transiently and long-term. Additionally, enhanced versions of BrecOFF with alternative KRAB domains were developed, and virus-like particles (VLPs) were engineered for efficient delivery. Results: Both dBrec1 and BrecOFF bound to the HIV LTR without inducing proviral cleavage. Transient expression of dBrec1 or BrecOFF repressed episomal HIV transcription, but only BrecOFF inhibited basal proviral transcription for over a month, indicating durable epigenomic silencing. Mutational analyses confirmed that DNA and histone methylation are essential for sustained repression. We further enhanced transcriptional silencing by substituting the KOX1 KRAB domain with ZIM2 or ZIM3. Finally, VLPs were validated as an efficient packaging and delivery system for BrecOFF, opening new opportunities to silence HIV in T cells. Conclusions: BrecOFF represents a promising HIV-specific epigenome editor with significant therapeutic potential. Combining dBrec1’s HIV-specific targeting with the durable epigenomic silencing of CRISPRoff, BrecOFF offers an innovative next-generation gene therapy strategy for a functional HIV cure.

The development of the VLP delivery system further supports its translational feasibility for both research and clinical applications. RV550: Safety and Virological Outcomes in Blood and Lymph Nodes of N-803 With ART in Acute Infection Carlo Sacdalan 1 , Eugène Kroon 1 , Meera Bose 2 , Shida Shangguan 2 , Nicole Dear 2 , Pathariya Promsena 1 , Nicha Tulmethakaan 1 , Anchalee Avihingsanon 3 , Jeffrey Safrit 4 , Somchai Sriplienchan 1 , Morgane Rolland 2 , Lydie Trautmann 5 , Kiat Ruxrungtham 6 , Sandhya Vasan 5 , for the RV550 Study Group 1 SEARCH, Bangkok, Thailand, 2 US Military HIV Research Program, Bethesda, MD, USA, 3 HIV-NAT, Thai Red Cross AIDS and Infectious Disease Research Centre, Bangkok, Thailand, 4 ImmunityBio, San Diego, CA, USA, 5 Henry M Jackson Foundation, Bethesda, MD, USA, 6 Thai Red Cross AIDS Research Centre, Bangkok, Thailand Background: We conducted a Phase 2, open-label study to evaluate the safety of N-803 with ART in acute HIV infection (AHI) and evaluated the impact on HIV viral load (VL), levels of vDNA and vRNA in CD4 T cells and lymph nodes (LN). Methods: We randomized 12 participants in AHI to receive immediate ART (n=4, Fiebig 1-2) or ART + N-803 (n=8, Fiebig 1-4) at 6 mcg/kg SC abdominally at weeks 0, 3, and 6 then followed to week 12. All participants had inguinal LN biopsy at wk 0 and week 6.5. Blood was drawn for VL, CD4 and CD8 count. Adverse events (AEs) were solicited every N-803 injection. CD4 T cells and LN vRNA and vDNA were compared using nonparametric Wilcoxon signed rank, Mann-Whitney U and Kruskal-Wallis tests and Spearman correlations. Results: Median (IQR) age was 32(26-35) years in the N-803 arm, 24(21-28) years in the controls. All N-803 recipients reported injection site redness at all time-points (n=24) with 4 mild, 3 moderate and 17 severe. For injection site swelling (n=23), 6 were mild, 9 moderate and 8 severe. Despite this, injection site AEs resolved in 7 days and all participants consented to continued N-803 administration. There was no significant difference at baseline for CD4 (300 vs 548 cells/mm 3 ), HIV VL (6.15 vs 5.73 log 10 ) and CD4 count increase over time between groups. While VL were overall comparable across groups, VL significantly declined (p=0.008) after the first dose of N-803 compared to ART-only (FigA). vRNA and vDNA were obtained from CD4 T cells and LN from 7 ART + N-803 participants and 4 ART participants. vRNA (LTR-Gag, Tat-Rev) and vDNA (total, integrated, 2-LTR circles) levels declined for all participants in CD4 T cells in 12 weeks and LN in 6.5 weeks. There was no significant difference in the decline of vRNA and vDNA between groups for LN (adjusted p≥0.545) and CD4 T cells (adjusted p≥0.218) from baseline (FigB). Conclusions: N-803 with ART was safe and resulted in a rapid plasma HIV VL decline from study baseline compared to ART alone. While there was some evidence of a faster decline in viremia immediately post N-803 injection, differences in reservoir measurements were overwhelmed by the large fluctuations that occur over the first weeks of ART in acute infection, inter-host variability, and the small number of participants. To further explore the impact of N-803 on viral reservoirs, all eligible and consenting study participants will receive an additional single dose of N-803 followed by ATI until HIV viral load >1,000 copies/ml for a maximum of 4 weeks. HIV-1 Reservoir Decay During Broadly Neutralizing Antibody Therapy in the RIO Trial Marcilio Jorge Fumagalli 1 , Anna Kaczynska 1 , Marius Allombert 1 , Agata Lopes Ribeiro 1 , Mariangela Silva 1 , Brianna Hernandez 1 , Christy L. Lavine 2 , Cintia Bittar 1 , Thiago Y. K. Oliveira 1 , Anna Gazumyan 1 , Michael S. Seaman 2 , John Frater 3 , Sarah Fidler 4 , Marina Caskey 1 , Michel Nussenzweig 1 , for the RIO Trial Investigators 1 The Rockefeller University, New York, NY, USA, 2 Beth Israel Deaconess Medical Center, Boston, MA, USA, 3 University of Oxford, Oxford, UK, 4 Imperial College London, London, UK Background: Antiretroviral therapy fails to eliminate a reservoir of HIV-1 infected cells wherein the provirus is transcribed at levels insufficient to induce cell death. Earlier studies of broadly neutralizing antibodies (bNAbs) administered during ART interruption (ATI) showed mixed results with some reporting accelerated reservoir decay. RIO enabled examination of reservoir dynamics in 32 individuals receiving 3BNC117-LS and 10-1074-LS during ATI or after ART restart. Methods: Participants were randomized 1:1 to bNAbs or placebo and underwent ATI. After initial rebound, the placebo group received bNAbs at ART restart. Intact and defective reservoirs were measured by digital droplet PCR (ddPCR) and by full genome sequencing (Q4PCR) at baseline (before ATI) and The figure, table, or graphic for this abstract has been removed.

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