ISSN: 2055-6640 Abstracts of the Eleventh International Workshop on HIV Persistence during Therapy December 10 to 13, 2024 Fort Lauderdale, Florida, USA Volume 10, Supplement December 2024
Journal of Virus Eradication Volume 8, Supplement Publication of this supplement is supported by amfAR. Abstracts of the Tenth International Workshop on HIV Persistence during Therapy December 13 to 16, 2022 Miami, Florida, USA Volume 10, Supplement December 10 to 13, 2024 Fort Laurderdale, Florida, USA Abstracts of the 11th International Workshop
Editor in Chief Sabine Kinloch-de Loës University College London Division of Infection and Immunity, London, United Kingdom Journal of Virus Eradication Editors Margaret Johnson Royal Free London NHS Foundation Trust, United Kingdom Christina K. Psomas European Hospital Marseille, Department of Infectious Diseases and Internal Medicine, Marseille, France Editorial Board Members Henry L.Y. Chan Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong Nicolas Chomont Vaccine and Gene Therapy Institute of Florida, Florida, United States Steven Deeks University of California San Francisco, San Francisco, United States Sarah Fidler St Mary’s University Faculty of Education, Humanities and Social Sciences, Twickenham, United Kingdom Paul Griffiths University College London Medical School, London, United Kingdom Alain Lafeuillade Intermunicipal Hospital Centre Toulon - La Seyne sur Mer, Toulon, France Seng Gee Lim Division of Gastroenterology and Hepatology National University Health System, Singapore Nelson Michael US Military HIV Research Program, Silver Spring, Maryland, United States Jean-Michel Pawlotsky Department of Biology and Pathology, Henri Mondor University Hospital, Créteil, France Irini Sereti National Institute of Allergy and Infectious Diseases, Bethesda, United States Janet Siliciano Johns Hopkins University School of Medicine, Baltimore, United States Robert F. Siliciano Johns Hopkins University School of Medicine, Baltimore, United States Guido Silvestri Emory University, Atlanta, United States Linos Vandekerckhove Ghent University, HIV Cure Research Center, Gent, Belgium Editorial Panel Juan Ambrosioni HIV Unit, Infectious Diseases Service Hospital Clinic-IDIBAPS, University of Barcelona, Barcelona, Spain David Asboe Chelsea and Westminster Hospital NHS Foundation Trust, United Kingdom Georg Behrens Hannover Medical School, Hannover, Germany Monsef Benkirane Laboratory of Molecular Virology, Montpellier, France Michael R. Betts University of Pennsylvania, Perelman School of Medicine, Penn Institute for Immunology, Philadelphia, United States Debika Bhattacharya Division of Infectious Diseases, University of California Los Angeles, Los Angeles, USA Simon Collins HIV i-Base, London, UK Zeger Debyser KU Leuven, Leuven, Belgium Jean-Francois Delfraissy National Agency for Research on AIDS and Viral Hepatitis, Paris, France Lucy Dorrell University of Oxford, Nuffield Department of Medicine, Oxford, United Kingdom Daniel Douek National Institute of Allergy and Infectious Diseases, Bethesda, United States Manal H El-Sayed Pediatric Department, Faculty of Medicine, Ain Shams University, Egypt Caroline Foster Imperial College Healthcare NHS Trust, London, United Kingdom Graham Foster Queen Mary University of London, London, United Kingdom John Frater University of Oxford, Oxford, United Kingdom Brian Gazzard Chelsea and Westminster Hospital NHS Foundation Trust, United Kingdom Anna Maria Geretti University of Liverpool, Liverpool, United Kingdom Carlo Giaquinto University of Padua, Padova, Italy George Hanna Princeton, New Jersey, United States Daria Hazuda Merck and Co Inc Global Human Health Business Development, North Wales, United States
Andrew Hill University of Liverpool, Liverpool, United Kingdom Ilesh Jani National Institute of Health Mozambique, Marracuene, Mozambique Rowena Johnston Foundation for AIDS Research, New York, United States Jerome Kim US Military HIV Research Program, Silver Spring, United States Richard Koup National Institute of Allergy and Infectious Diseases, Immunology Laboratory, Bethesda, United States Nagalingeswaran Kumarasamy YR Gaitonde Centre for AIDS Research and Education, Chennai, India Alan Landay Rush University Medical Center, Chicago, United States Clifford Leen Western General Hospital, Edinburgh, United Kingdom Yves Levy National Institute of Health and Medical Research, Paris, France Sharon Lewin The University of Melbourne Asialink, Parkville, Australia Carine van Lint ULB Institute of Molecular Biology and Medicine, Gosselies, Belgium Jan Van Lunzen University Medical Center Hamburg-Eppendorf, Hamburg, Germany Hermione Lyall St Mary’s Hospital, London, United Kingdom Giulia Marchetti Department of Health Sciences, Clinic of Infectious Diseases, University of Milan, Milan, Italy Gail Matthews University of New South Wales, Sydney, Australia Veronica Miller The Forum for Collaborative Research, Washington, United States Piotr Nowak Department of Medicine; Unit of Infectious Diseases, Karolinska Institute, Stockholm, Sweden Cristiana Oprea Victor Babes Clinical Hospital for Infectious and Tropical Diseases, Bucureşti, Romania Melanie Ott Gladstone Institute of Virology and Immunology, San Francisco, United States Carlo-Federico Perno University of Rome Tor Vergata, Roma, Italy Nittaya Phanuphak Thai Red Cross AIDS Research Centre, Bangkok, Thailand Guido Poli University Life-Health Saint Raphael, School of Medicine, Milano, Italy Sarah Read National Institute of Allergy and Infectious Diseases, Bethesda, United States Doug Richman University of California San Diego, La Jolla, United States Christine Rouzioux Necker Hospital in Paris, France Pedro Simões Royal Free Hospital NHS Foundation Trust, North Middlesex University Hospital NHS Trust, UK Serena Spudich Yale University School of Medicine, New Haven, United States Claire Thorne University College London, London, United Kingdom Gilles Wandeler Department of Infectious Diseases, Bern University Hospital, Bern, Switzerland
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Abstracts of the 11th International Workshop on HIV Persistence during Therapy Volume 10 Contents Oral Presentations Session 1 Basic Science of HIV Persistence 1 Session 2 Virology of HIV Persistence 4 Session 3 Drug Discovery & Development, Pharmacology, Novel approaches 7 Session 4 Immunology of HIV Persistence 12 Session 5 In Vitro and Animal Model Studies of HIV Persistence 16 Session 6 Cell & Gene Therapies 20 Session 7 Human Studies 24 Session 8 Antibody & Immune based Therapies 28 Highlighted Short Talks of Interest I 32 Highlighted Short Talks of Interest II 34 Poster Presentations Poster Presentations Basic Science of HIV Persistence 38 Poster Presentations Virology of HIV Persistence 51 Poster Presentations Drug Discovery & Development, Pharmacology, Novel approaches 57 Poster Presentations Immunology of HIV Persistence 62 Poster Presentations In Vitro and Animal Model Studies of HIV Persistence 72 Poster Presentations Cell and Gene Therapies 78 Poster Presentations Human Studies 82 Poster Presentations Antibody & Immune based Therapies 89
Abstracts of the Eleventh International Workshop on HIV Persistence during Therapy Oral Presentations Session 1: Basic Science of HIV Persistence 1.1– 00003 Longitudinal analysis in early treated individuals reveals alteration in the HIV-1 integration site landscape and composition of the inducible reservoir T. Struyve1,*, M. Pardons1, J. De Clercq1, L. Termote1, L. Lambrechts1, Y. Noppe1, M. Lichterfeld2, S. Rutsaert1, L. Vandekerckhove1 1HIV Cure Research Center, Department of Internal Medicine and Pediatrics, Ghent University Hospital, Ghent University - Ghent, Belgium; 2Infectious Disease Division, Brigham and Women’s Hospital, Ragon Institute of MGH, MIT and Harvard, Boston, United States Background/Aims: Little is known about clonality, integration site (IS) features and inducibility in early treated individuals. Therefore, we performed a longitudinal analysis of the HIV-1 viral reservoir in this population. Methods: We included eight individuals who started treatment during acute infection and collected blood samples after a median of 0.7 years (Acute undetectable (UD)) and 5.3 years (Acute UD+5) on antiretroviral therapy (ART). For comparison, a cohort of individuals who initiated ART during chronic infection (Chronic UD) was included. IS were obtained by Integration Site Loop Amplification. Following a 24h-stimulation with PMA and an HIV-1 Tat mRNA-containing nanoparticle (TatLNP), the frequency and phenotype of p24-expressing cells were assessed by HIV-Flow. Results: Clonally infected cells were retrieved in only 4/8 participants from the Acute UD cohort (n = 452 IS), while all individuals from the Chronic UD cohort (n = 197 IS) displayed multiple distinct IS detected more than once. The proportion of IS detected more than once significantly increased from 2.9% for Acute UD to 12.9% for Acute UD+5 (n = 124 IS) (p < 0.0001). Further characterization revealed similar IS features between the Acute and Chronic cohorts at UD regarding (i) orientation of proviruses in genic regions, (ii) proportions of IS into ZNF genes with KRAB domain and centromeric DNA, (iii) integration patterns in repeat regions and Hi-C compartments. In the Acute cohort, proportions of proviruses in centromeric DNA increased from 0.9% to 8.9% after 5 years on ART (p < 0.0001), while there was a significant decline in proviruses located in proximity to activating histone marks (H3K4me1, H3K4me3, H3K27ac; p < 0.05 for all). Following PMA/Tat-LNP stimulation, the proportion of infected cells with an inducible provirus was similar in the Acute and Chronic cohorts. The proportion of p24 + cells with a naïve phenotype decreased over time on ART in both cohorts, while the majority of p24+ cells displayed an effector memory phenotype at the later timepoint. Conclusion: Early after ART initiation, IS features and inducibility were similar irrespective of treatment initiation timing. Our findings suggest that in early treated individuals host selection mechanisms can already reshape the viral reservoir landscape as early as 5 years after ART initiation. Any conflict of interests: NA. Journal of Virus Eradication 10S (2024) 100394 https://10.1016/j.jve.2024.100394 1.2– 00055 Blood and tissue HIV-1 reservoirs display cellular plasticity and lack of compartmentalization in virally suppressed people M.Pardons1,*, L. Lambrechts1,Y.Noppe1, L. Termote1, S. De Braekeleer1, J. Vega2, E. Van Gulck3, S. Gerlo1, L. Vandekerckhove1 1Hiv Cure Research Center - Ghent, Belgium; 2Arcturus Therapeutics, San Diego, United States; 3Janssen Pharmaceutica Nv - Beerse, Belgium Background/Aims: Characterizing the HIV-1 reservoir in blood and tissues is key to advance cure interventions. We aimed to address whether the inducible reservoirs in matched blood and lymph nodes (LN) exhibit distinct phenotypic features and if near full-length (NFL) sequencing reveals compartmentalization between blood and tissues. Methods: Matched blood, LN and/or gut biopsies were obtained from 9 HIV+ individuals undergoing treatment for an average time of 14.6y (T1). For 6/9 participants, blood from a previous time point was available (T0). Following a 24hstimulation with a Tat mRNA-containing lipid nanoparticle (Tat-LNP) and panobinostat (PNB), phenotypic characterization of blood (n = 128) and LN (n = 55) p24+ cells was performed using HIV-Flow (T1). NFL sequencing (n = 3421 proviral genomes) was conducted using HIV Proviral UMI-mediated Long-read Sequencing on blood (T0/T1), LN and gut (T1). Results: Following Tat-LNP/PNB stimulation, the p24+ cell frequencies were similar between blood and LN (means = 11.5 and 9.0 p24+ cells/106 CD4 T cells), suggesting comparable sizes of the inducible reservoirs in both compartments. P24+ cells from blood and LN displayed distinct phenotypes. Bloodderived p24+ cells were found in both central/transitional (TCM/TTM) and effector memory subsets, with only 8.6% expressing the follicular homing marker CXCR5 and 17.2% expressing the cytotoxic GZMA molecule. In contrast, most p24+ Journal of Virus Eradication 10S (2024) Abstracts Contents lists available at ScienceDirect Journal of Virus Eradication journal homepage: www.sciencedirect.com/journal/journal-of-virus-eradication 2055-6640/ © 2024 Published by Elsevier B.V
cells from lymph nodes exhibited a TCM/TTM phenotype, with 53% expressing CXCR5 and only 1.8% expressing GZMA. Furthermore, none of the LN p24+ cells exhibited a germinal center T follicular helper (GC Tfh) phenotype (CXCR5highPD1high). Average pairwise distance calculations, panmixia testing and phylogenetic analyses all indicated a lack of compartmentalization between blood and tissues. We detected 175 overlapping NFL sequences between time points and/or anatomical sites, including intact and inducible sequences, indicating that clones of infected cells can persist over time and disseminate through the body using the blood as a conduit. Conclusion: Our study suggests that clones of infected cells may display distinct phenotypes based on their anatomical location, highlighting the plasticity of infected immune cells in response to their microenvironment. Furthermore, our findings indicate that GC Tfh cells may not be preferential targets for cure interventions in long-term treated individuals. Any conflict of interests: The Authors MP, LL, YN, LT, SDB, SG and LV declare no competing interests. The Author EVG was an employee of Johnson & Johnson and may be Johnson & Johnson stockholder. The Author JV was an employee of Arcturus Therapeutics and may be Arcturus Therapeutics stockholder. Journal of Virus Eradication 10S (2024) 100395 https://10.1016/j.jve.2024.100395 1.3– 00084 Persistence of HIV genomes in bacteria-specific CD4+ T cells during ART A. Espinosa Ortiz1,2,*, R. Fromentin1,2, S. G. Deeks3, J.P. Routy4, N. Chomont1,2 1Département De Microbiologie, Infectiologie Et Immunologie, Faculté De Médecine, Université De Montréal - Montréal, Canada; 2Centre De Recherche Du Chum - Montréal; 3Department of Medicine, University of California, San Francisco, California, United States; 4Division of Hematology & Chronic Viral Illness Service, McGill University Health Centre - Montréal, Canada Background/Aims: Clonal expansions of infected cells contribute to the maintenance of HIV reservoirs during anti-retroviral therapy (ART). However, the antigen specificities of these T cells clones remain elusive. Since intestinal CD4+ T cells are highly susceptible to HIV infection, we hypothesized that gut bacteriaresponsive CD4+ T cells may be preferential targets for HIV and contribute to HIV persistence through their proliferation. Methods: To determine the frequency of bacteria-responsive CD4+ T cells, we stimulated PBMCs from 10 HIV negative, 10 ART-suppressed and 10 viremic individuals with bacterial lysates from 13 bacteria representing the main phyla of the gut microbiota (including B. animalis, C. difficile, E. coli, L. acidophilus S. typhimurium and P. stercorea). We used the combined expression of 3 activation markers (CD69, 4–1BB and CD40L) to detect bacteria-specific CD4+ T cells (AIM+) in the blood. To evaluate the contribution of gut bacteria-responsive CD4+ T cells to the HIV reservoir, we sorted AIM+ cells from virally suppressed individuals and sequenced near-full length (NFL) HIV genomes using a modified FLIPS assay. Results: Bacteria-responsive CD4+ T cells were detected at significantly lower frequencies in viremic individuals compared to uninfected controls (mean frequencies of 0.05% and 0.17%, p < 0.001), and at intermediate levels in people suppressed on ART (0.11%). We retrieved 111 NFL-proviral sequences from bacteria-responsive and total CD4+ T cells from 3 ARTsuppressed participants. All proviruses were defective, except for 2 genetically intact proviruses detected in S. typhimuriumresponsive CD4+ T cells. Interestingly, clonally expanded proviruses were frequently detected in microbiota-responsive CD4+ T cells (56% clonality compared to 8% in total CD4+ T cells). Conclusion: Our results suggest that microbiota-reactive CD4+ T cells may be preferentially depleted during active HIV infection and not entirely restored by ART. These cells harbour clonally expanded proviruses indicating that their proliferation contribute to HIV persistence, including genetically intact genomes. Altogether, these findings identify bacteria-specific CD4+ T cells as a novel reservoir for HIV. Any conflict of interests: Absence of conflict of interests to disclose. Journal of Virus Eradication 10S (2024) 100396 https://10.1016/j.jve.2024.100396 1.5– 00118 Intact Proviruses Persist in Expressed Genes in People with HIV-1 on Long-term ART S. Patro1,*, J. Gluck2, E. Halvas3, K. Joseph3, N. Mckenna3, S. Guo1, S. Parvez2, J. Rausch2, X.Wu1, J. Mellors3, S. Hughes2, M. Kearney2 1Leidos Biomedical Research, Inc., Frederick National Laboratory for Cancer Research - Frederick, MD, United States; 2National Cancer Institute - Frederick, MD, United States; 3University of Pittsburgh - Pittsburgh, PA, United States Background/Aims: The reservoir of intact proviruses that persist despite ART has been incompletely characterized. By studying thousands of infected cells that persist on long-term ART, we asked, “How many infected CD4+ T cell clones harbor intact proviruses? Do these clones grow larger or smaller over time? How often are the intact proviruses integrated into genes? Are these genes expressed? Are the proviruses inducible and the source of non-suppressible plasma viremia on ART?”. Methods: Three adult PWH on ART for 10–20 years were sampled over 1–4 years. Genomic DNA from PBMC underwent endpoint-dilution multiple displacement amplification (MDA). MDA wells were screened by qPCR for HIV LTR, psi (Ψ), andRRE. Proviruses positive for all three underwent full-length HIV sequencing and integration sites analysis. Integration sites were mapped to single or multiple copy host sequences (e.g., satellite). RNA levels of integration site-associated genes were determined using RNA-seq of autologous CD4+ T cells. Sub-genomic regions of intact proviruses were matched to viral RNA in plasma and outgrowth virus. Results: We identified 11 cell clones carrying intact proviruses confirmed by ProSeq-IT analysis. The integration sites of the majority (10/11) mapped to genic introns. Only 1 provirus mapped to a satellite repeat, HSat3B5 (chr9). Six of the intact proviruses were in KRAB-ZNF genes (p < 3.4 × 10–8 vs all proviruses), and the same 6 were within 1% of the end of a chromosome. Four of the proviruses were in the same orientation as the gene. mRNA levels of the 10 genes in which integrations occurred varied widely (96.9–1.91 TPM). Over 1–4 years, 2 of the clones increased in size, 1 decreased, and 7 were stable. SubAbstracts Journal of Virus Eradication 10S (2024) 2
genomic regions of all intact proviruses matched virus in either viral outgrowth assays or plasma viremia. Conclusion: We characterized 11 clones carrying intact proviruses in 3 PWH on long-term ART. Most clones were stable; only 2 decreased in size. Almost all intact proviruses were integrated in genes (10 of 11) and 6 of 10 were in KRAB-ZNF genes near the ends of chromosomes. These data show persistence of intact proviruses in limited genic locations that could be specifically targeted for silencing or elimination. Any conflict of interests: JWM is a consultant to Gilead Sciences and grant recipient from Gilead Sciences to University of Pittsburgh. Journal of Virus Eradication 10S (2024) 100397 https://10.1016/j.jve.2024.100397 1.6– 00161 HIV-1 Antisense Transcripts are frequent in FOXP3-negative Treglike cells expressing markers of persistence in vivo during acute HIV1 infection M. Hale1,2,*, G. Kundu1,2, A. Geretz1,2, P. Ehrenberg1, R. Clifford1,2, M. Robb1,2, C. Sacdalan3,4, S. Sriplienchan3,4, N. Phanuphak5, S. Vasan1,2, R. Thomas1, RV254 study team 1US Military HIV Research Program, Walter Reed Army Institute of Research - Silver Spring, MD, United States; 2Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. - Bethesda, MD, United States; 3SEARCH Research Foundation - Bangkok, Thailand; 4Research Affairs, Faculty of Medicine, Chulalongkorn University - Bangkok, Thailand; 5Institute of HIV Research and Innovation - Bangkok, Thailand Background/Aims: The majority of HIV-1 coding sequences, including those necessary for replication, occur on the forward, or sense, strand of the HIV-1 genome. Despite this, the HIV-1 antisense strand contains a single ORF which encodes the Antisense Protein (asp). This gene sits antisense to env and is bifunctional, acting as a structural component of the HIV-1 virion during active infection and to suppress viral replication as a lncRNA. However, despite its predicted relevance to infectivity, our understanding of asp and expression of its transcript (AST) remains limited in vivo during acute HIV infection (AHI). Methods: Droplet-based single-cell RNA sequencing (scRNAseq) was performed in CD4+ memory T-cells to identify host and viral transcripts for 4 Thai participants from AHI cohorts. To characterize host gene expression patterns associated with HIV-1 antisense transcription, including AST, we used the bioinformatic packages ‘MAST’ and ‘Seurat’ to compare host gene expression within HIV-1 viral RNA positive (vRNA+) cells expressing antisense transcripts and vRNA+ cells that did not express detectable antisense transcript. Results: HIV-1 vRNA+ cells expressing AST were detected in all participants during AHI (< 6% of total vRNA+ cells). Antisense reads from beyond the asp locus were also detected, and we observed strong positive correlations between antisense and total sense expression within vRNA+ cells, including AST. Clustering analysis within vRNA+ cells identified two subclusters enriched for expression of AST; marker gene expression in these two groups suggested they may be FOXP3-negative Treglike populations. Markers of persistence, including TIGIT, CTLA4, and PD-1, were expressed at higher levels in these clusters. Direct gene expression comparisons between AST+ and ASTvRNA+ cells identified 417 distinct differentially expressed genes, including 230 genes expressed at significantly higher levels in AST+ cells, some of which likely reflect differences between cell types of AST+ cells. Conclusion: The HIV-1 antisense strand is actively transcribed during AHI and is associated with distinct patterns of host gene expression within infected cells. Moreover, Treg-like cells may be predisposed to express AST, and additional work to validate these patterns and to assess associations with clinical outcomes and immunological parameters are ongoing. Any conflict of interests: The authors have no conflicts of interest. Figure (abstract: 1.5– 00118). Abstracts Journal of Virus Eradication 10S (2024) 3
Journal of Virus Eradication 10S (2024) 100398 https://10.1016/j.jve.2024.100398 Session 2: Virology of HIV Persistence 2.1– 00040 Lenacapavir impairs gag proteins expression by HIV-infected cells C. Faua1,*, S. Bernacchi2, A. Ursenbach3, M. Negroni2, P. Gantner1,4 1INSERM UMR_S1109, Strasbourg University, Strasbourg, France; 2Architecture et Réactivité de l’ARN-UPR 9002, IBMC, CNRS, Université de Strasbourg, Strasbourg, France; 3Le Trait d’Union, HIV-Infection Care Center, Strasbourg University Hospital, Strasbourg, France; 4Clinical Virology Laboratory, Strasbourg University Hospital, Strasbourg, France Background/Aims: Due to its high binding affinity to HIV capsid, the first-in-class capsid inhibitor Lenacapavir (LEN) might display a post-integration effect on capsid production. However, this effect and its underlying mechanisms have yet to be characterized. Methods: The post-integration effect of increasing concentrations of LEN was measured in vitro (8E5, ACH2, U1, transfected 293T cells) and ex vivo (PBMCs from n = 10 viremic HIVinfected participants enrolled in the APRIL study [NCT05752318]) by analysing HIV transcripts and proteins expression by flow cytometry and p24 and HIV-RNA levels in supernatants. Results: As soon as 15 minutes of exposure, LEN induces a rapid dose-dependent intracellular capsid (p24) expression decrease at therapeutic concentrations (IC50 = 10 nM) that reaches top plateau with 50 nM of LEN in HIV-1 infected cell lines, whereas HIV-2 infected cells were less sensitive to LEN. For the latently-infected cell line (U1), this decreased p24 expression on LEN occurred independently of the activation pathway targeted. Whereas extracellular envelope protein expression was not affected by LEN, intracellular matrix (p17) proteins expression was also reduced with the same magnitude as p24. These decreases further translated in lower levels of viral particle release on LEN. Intracellular HIV gag transcripts levels were unchanged by LEN and maturation inhibition did not affect the decreased p24 expression induced by LEN, suggesting a post-transcription but pre-maturation effect. p24 expression by oligomerization mutants (W184D +M185D, P38E+A42E+E45R) and LEN-resistant mutants (N74D and Q67H+N74D) were less affected by LEN, suggesting that a specific binding to capsid oligomers is required. LEN also impaired p24 expression ex vivo in productively-infected CD4+ T cells from viremic HIV-infected individuals (decreased intracellular p24 [but not envelope] expression and decreased viral particle release). Conclusion: Altogether, LEN induces a rapid and specific decrease of not only capsid but also other gag proteins in vitro and ex vivo by HIV-infected cells. Reduced gag proteins expression by reservoir cells in LEN-treated individuals could impact recognition of these cells by the immune system and thus the dynamics of HIV reservoirs and should be further studied. Any Conflict of Interests: The authors declare no conflict of interests. Journal of Virus Eradication 10S (2024) 100399 https://10.1016/j.jve.2024.100399 2.2– 00058 Role of HIV integration site on clonal expansion of infected cells and maintenance of latency in vivo V. Pal1,*, M. Frauke2, A. Danesh3,M. Canis1, T. Dilling3, I.Miller3, T. Huynh3, T. Hatziioannou1, R.B. Jones3, G.Q. Lee3, P.D. Bieniasz1,4 1The Rockefeller University - New York, United States; 2Heidelberg University - Heidelberg, Germany; 3Weill Cornell Medical College - New York, United States; 4Howard Hughes Medical Institute, The Rockefeller University, New York, United States Background/Aims: Latent reservoirs of HIV-1 are the major barrier to cure. Because latently infected cells are rare, there is a paucity of understanding of how 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 clones during ART. In this study, we characterized the proviral IS associated with clonal expansion and latency of HIV-1 infected cells in an in vivo model. Methods: Memory CD4+ T cells isolated from healthy human donors were infected with dual reporter- tagged, defective HIV-1 to generate large populations (millions) of infected cells, each presumptively carrying a single transcriptionally active reporter provirus at a distinct IS. These cells were grafted into NSG mice and analyzed for proviral reporter expression dynamics. HIV-1 IS were determined by PCR-based amplification of host-viral junction and next-generation sequencing. Results: Over time, the fraction of engrafted cells in which the HIV-1 reporter provirus was transcriptionally active decreased. HIV-1 IS analysis in long-term persistently infected cell clones led to the identification of 646 unique IS. Consistent with previous reports, HIV-1 integration was favored in genic regions (80%) of human chromosomes as compared to non-genic regions (20%). HIV-1 integrations were primarily in introns (95%). Comparison of genic IS in multiple animals lead 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. Sorting of cells with active and latent proviruses at later time points after engraftment revealed that IS harboring transcriptionally active proviruses were more likely to be found within genes compared to those harboring transcriptionally latent proviruses. Strikingly, IS harboring the majority of latent proviruses were enriched in non-genic, pericentric regions of human chromosomes. Conclusion: HIV-1 IS affects proviral transcriptional status, and non-genic IS more frequently harbor latent proviruses months after engraftment of transcriptionally active proviruscontaining cells into animals. Current and future works involves studying the mechanisms underpinning individual IS-driven clonal survival of infected cells and proviral transcriptional status. Any Conflict of Interests: No Conflict of interest. Abstracts Journal of Virus Eradication 10S (2024) 4
Journal of Virus Eradication 10S (2024) 100400 https://10.1016/j.jve.2024.100400 2.3– 00078 Detection of HIV-1 antisense transcripts in donor samples before and during ART A. Capoferri1,*, T.O. Famuyiwa1, R. Sklutuis1, S. Pathak1, J.L. Groebner1, R.Li2, J.W. Rausch1, S.G. Deeks3, J.W. Mellors4, J.M. Coffin5, F. Romerio2, M.F. Kearney1 1HIV Dynamics and Replication Program, National Cancer Institute, Frederick, MD, United States; 2Department of Molecular and Comparative Pathobiology, The Johns Hopkins University School of Medicine, Baltimore, MD, United States; 3Department of Medicine, University of California, San Francisco, CA, United States; 4Department of Infectious Diseases, University of Pittsburgh, Pittsburgh, PA, United States; 5Department of Molecular Biology and Microbiology, Tufts University, Boston, MA, United States Background/Aims: Natural antisense transcripts (AST) are expressed by viruses, prokaryotes, and eukaryotes and primarily function to regulate sense gene expression. In vitro studies have shown that HIV-1 expresses AST from a Tat-independent promoter within the 3′ LTR and promotes latency through epigenetic modification of nucleosomes at the 5′ LTR by the PRC2-EZH2-H3K27me3 pathway. We asked if HIV-1 AST are expressed in PBMC from PWH before and during ART. Methods: PBMC were isolated from 8 PWH. One donor provided 4 longitudinal samples, 2 before an ART interruption and 2 shortly after ART re-initiation. Five samples were from donors not on ART (plasma HIV-1 RNA 128–275,000 copies/ml) and 6 on ART (<50–112 copies/ml). AST levels were measured by cell-associated antisense RNA single-genome sequencing and by endpoint digital PCR assays using donor-specific primers. In 2 of the donors on ART, env single-genome sequences from PBMC and lymph node mononuclear cells (LNMC) were also obtained. Results: HIV-1 AST was detected in all samples. The median level of AST in samples not on ART was 2 copies/100 HIV-1 DNA + cells [IQR 1–19] and 26 copies/100 HIV-1 DNA+ cells [IQR 16–47] in samples on ART (p = 0.05). Higher values were seen in samples collected weeks after ART re-initiation compared to long-term ART (49 vs. 25, p = 3.2 × 10−9). Sequencing of AST revealed high genetic diversity ranging from 0.7–2.5%. AST sequences matched a diverse population of proviruses in PBMC and LNMC, indicating that a wide variety of proviruses can express AST. Conclusion: HIV-1 AST was detected in all PWH with 13-fold higher levels in samples from donors on ART than not on ART when normalized for HIV-1 DNA+ cells. These results are consistent with a potential role of AST in regulating HIV-1 sense gene expression and inducing latency. Further studies are required to establish the possible regulatory role of HIV-1 AST in strategies to silence HIV-1 transcription. Any Conflict of Interests: JWM is a consultant to Gilead Sciences, has received research grants from Gilead Sciences to the University of Pittsburgh, and owns share options in Infectious Disease Connect (co-founder) and Galapagos, NV, unrelated to the current work on HIV. JMC is a member of the Scientific Advisory Board and a Shareholder of ROME Therapeutics, Inc. and Generate Biomedicine, Inc. The remaining authors have no potential conflicts. Journal of Virus Eradication 10S (2024) 100401 https://10.1016/j.jve.2024.100401 2.4– 00143 Selective export of HIV mRNAs is regulated by compartmentalized interactions with Sam68, PTB and m6A RNA methylation in reactivated latently infected T-cells F. Kizito1, E. Honeycutt1, F. Ye1, T. Sweet2, A. Agaponova1, J. Karn1,* 1Department of Molecular Biology and Microbiology; 2Department of Nutrition, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106, USA Background/Aims: Background: Post-transcriptional processing and chemical modifications are essential for generating HIV mRNA diversity and determining the fate between nuclear retention and Rev-mediated export. These processes depend on host cellular cofactors to control the steps of alternative mRNA splicing and conserved internal N6-methyladenosine (m6A) regulation. Although, subnuclear compartmentalization and the significance of chemical modifications have been studied, little is known about the critical nuclear compartmentation and chemical modifications which determine the fate between nuclear retention and export of the early viral mRNA ribonucleoprotein complexes (RNPs), shortly after proviral reactivation from latency. Methods: The current study used in situ imaging analysis to track the spatiotemporal induction, nascent HIV-RNA subnuclear compartmentation, segregation, early RNP assembly, and export dynamics following proviral reactivation from latency. We developed highly selective FISH-RNA probes, distinguishing between completely spliced (msRNA) and intron-containing HIV-RNA (RRE-RNA) transcripts. Results: Using the primary QUECEL model of HIV latency, we show that upon proviral reactivation from latency, total HIV mRNA accumulated in the perinucleolar compartment (PNC) from where msRNAs were initially exported to the cytoplasm. In contrast, the export of RRE-RNA was delayed under retention in the PNC. The PNC-retained RRE-RNA was then segregated into two (2) portions: one portion trafficked to the nucleolus in a Revindependent manner and another PTB-mediated portion shuttled between the PNC and the nucleoplasm. The latter portion was strictly nuclear and strongly colocalized with Rev, DDX1, CRM1, and Sam68 from the PNC before trafficking to the nucleoplasm, where portions containing Rev, CRM1, and Sam68 subsequently segregated and trafficked to the cytoplasm. We hypothesized that the differential nuclear trafficking of mRNAs may be regulated by m6A modifications. In cells pretreated with METTL3 inhibitor STM-2457, the RRE-RNAs which are partially retained in the nucleus, were almost exclusively exported to the cytoplasm. Using RNA-Seq analysis, we mapped m6A modification sites of HIV-RNA, and computational analysis identified the binding motifs for PTBP1 and Sam68, which colocalized with the potential m6A modification sites in HIVRNA. Conclusion: Our results suggest that subnuclear compartmentation of HIV-RNA between the PNC and Sam68 nuclear bodies is a critical regulatory step of HIV-RNA, which may be mediated by m6A. Abstracts Journal of Virus Eradication 10S (2024) 5
Any Conflict of Interests: None. Journal of Virus Eradication 10S (2024) 100402 https://10.1016/j.jve.2024.100402 2.5– 00074 Propagation of HIV reservoir clones reveals functional heterogeneity, suggesting diverse mechanisms of persistence I. Ferreira1,*, A. Herrera1, T.T. Huynh1, E. Stone1, N. Linden1, C. Bittar Oliva2,M. Caskey2, M. Nussenzweig2, R.B. Jones1 1Division of Infectious Diseases, Weill Cornell Medicine, New York, USA; 2Laboratory of Molecular Immunology, The Rockefeller University, New York, USA Background/Aims: The reservoir of intact HIV proviruses that persists on ART is predominately harbored within clonally expanded CD4+ T-cells. Functional characteristics of both the cell and of the provirus/integration site likely contribute to persistence. However, the extreme scarcity of these cells ex vivo poses a barrier to their study. We report the expansion and characterization of ‘authentic reservoir clones’ (ARCs), revealing insights into persistence. Methods: ARCs were isolated from CD4+ memory T cells (n = 3) via rounds of limiting dilution with irradiated feeder cells, anti-CD3, anti-CD28, ARVs and bNAbs. At each round, wells containing expanded clones were detected using p24 ELISA. Proviruses were characterized by IPDA, near full-length sequencing, and integration site analysis. Clonality of ARCs was confirmed by TCR sequencing. Functional assays, including proliferation (n = 2), latency reversal (n = 2), and CTL-mediated killing susceptibility (n = 3), were analyzed via flow cytometry. Transcriptomic and immunophenotypic profiles were assessed with CITE-seq, and antigen specificities were determined by ELISPOT. Results: Each of the three ARCs harbored a single intact provirus integrated into either ZNF721, ATPB4, or ZNF486. The ZNF486 ARC was deeply latent, showing maximal reactivation of only 0.04 ± 0.3% Gag+ in response to anti-CD3/anti-CD28 (0% at baseline). ZNF721 ARC exhibited higher basal Gag expression (1.16 ± 1.18%) and was most responsive to bryostatin-1 (3.27 ± 1.22%), compared with 1.73 ± 1.22% for anti-CD3/antiCD28. CITE-seq revealed distinct transcriptional and phenotypic profiles, but with shared features of cytotoxicity and interferonstimulated gene expression. The ATPB4 ARC (peptide-pulsed) resisted CTL killing, while the ZNF486 ARC was highly susceptible. The ZNF721 ARC had a proliferative advantage over autologous uninfected CD4+ clones; purity was confirmed by IPDA, and EBV specificity was demonstrated. Conclusion: The ARCs displayed heterogeneous phenotypic, transcriptional, and functional traits suggesting differential reliance on three mechanisms of persistence. The ZNF721 ARC - which we have grown from 1 cell to >109cells - is distinguished by its enhanced proliferative capacity, whereas ZNF486 ARC is unique in its depth of latency. The ATPB4 ARC grows poorly and has the highest basal HIV expression but exhibits resistance to CTL-mediated killing. ARCs provide a tool for probing mechanisms underlying these features towards the goal of designing therapeutic strategies to eliminate reservoir clones. Any Conflict of Interests: No. Journal of Virus Eradication 10S (2024) 100403 https://10.1016/j.jve.2024.100403 2.6– 00158 Doubling dolutegravir dosage reduces the viral reservoir in ARTtreated people with HIV A. Pasternak1,*, C. Fombellida-Lopez2, A. Cicilionytė1, L. Winchester3, M.Maes4, P. Dellot5, C. Vanwinge6, A. Ladang7, E. Cavalier7, F. Susin8, D. Vaira8, M.P. Hayette8, C. Reenaers9, M. Moutschen2, C. Fletcher3, G. Darcis2 1Laboratory of Experimental Virology, Department of Medical Microbiology, Amsterdam Umc - Amsterdam, Netherlands; 2Laboratory of Immunology and Infectious Diseases, Giga-Institute, University of Liège - Liège, Belgium; 3Antiviral Pharmacology Laboratory, University of Nebraska Medical Center - Omaha, United States; 4Department of Biostatistics and Medico-Economic Information, University Hospital of Liège - Liège, Belgium; 5Department of General Internal Medicine and Infectious Diseases, University Hospital of Liège - Liège, Belgium; 6Giga Flow Cytometry Platform, University of Liège - Liège, Belgium; 7Department of Clinical Chemistry, University Hospital of Liège - Liège, Belgium; 8Laboratory of Clinical Microbiology, University Hospital of Liège - Liège, Belgium; 9Department of Gastroenterology, University Hospital of Liège - Liège, Belgium Background/Aims: Whether ART is always completely suppressive, or HIV might continue to replicate at low levels despite ART Figure (abstract: 2.5– 00074). Abstracts Journal of Virus Eradication 10S (2024) 6
in some people with HIV (PWH), is still debated. Here, we intensified the ART regimen by doubling dolutegravir (DTG) dosage and investigated the impact of this strategy on HIV blood and tissue reservoirs, immune activation, immune exhaustion, and inflammation. Methods: Twenty HIV-infected adults, who had received a triple ART consisting of 50 mg DTG/600 mg abacavir/300 mg lamivudine pre-intensification and had been suppressed on ART for at least two years, were enrolled in a phase 2 randomized clinical trial. Half of them received an additional 50 mg of DTG for a period of 84 days. We longitudinally quantified total HIV DNA in PBMCs and rectal tissue, intact HIV DNA in PBMCs, and cell-associated unspliced (US) HIV RNA in PBMCs. Expression of immune activation (HLA-DR, CD38) and exhaustion (PD-1, TIGIT) markers on peripheral CD4+ and CD8+ T cells was measured by flow cytometry. Systemic inflammation was assessed by measuring the levels of plasma levels of inflammatory cytokines IL-6, IL-1β, IL-17α, IFN-γ, and TNF-α. In rectal tissue, we measured expression of the same cytokines by quantifying their mRNA levels. Results: Plasma and tissue DTG concentrations significantly increased during the study period in the intensified group but not in the control group. Accordingly, significant longitudinal decreases in total HIV DNA, intact HIV DNA, and US HIV RNA in PBMCs, as well as in the US RNA/total DNA ratio, were observed in the intensified group but not in the control group. However, intensification did not affect total HIV DNA levels in rectal tissue. Intensification reduced markers of immune activation (% CD8+ T cells co-expressing CD38 and HLA-DR) and exhaustion (% CD4+ T cells expressing TIGIT) but had no measurable impact on systemic or tissue inflammation. Intensification also resulted in a temporary decrease in the CD4/CD8 ratio that returned to baseline by day 84. Conclusion: Our results strongly suggest that the preintensification ART regimen was not completely suppressive. If confirmed in larger clinical trials, these results could have an impact on the clinical management of PWH and HIV curative strategies. Any Conflict of Interests: None. Journal of Virus Eradication 10S (2024) 100404 https://10.1016/j.jve.2024.100404 Session 3: Drug Discovery & Development, Pharmacology, Novel approaches 3.1– 00145 Exploring novel HIV Tat inhibitors S.M. Jablonski1, J.A. Jablonski1, L. Shuang1, L. Ling2, A. T. McAuley1, R. Ronald, Jr.3, P. Espinoza-Gonzales1, B. MacTavish1, Q. Gibault1, S. Zhang4, T. Bannister3, S. M. Schader5, R. Ptak4, V. Garcia2, C. Augelli-Szafran4, S. T. Valente1 1Department of Immunology and Microbiology, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology, Jupiter, Florida, USA; 2Department of Microbiology, The University of Alabama, Birmingham, Alabama, USA; 3Department of Molecular Medicine, The Herbert Wertheim UF Scripps Institute for Biomedical Innovation & Technology, 130 Scripps Way, 2A1, Jupiter, Florida, USA; 4Drug Discovery Division, Chemistry Department, Southern Research, Birmingham, Alabama, USA; 5ViiV Healthcare, 410 Blackwell Street, Durham, NC 27701, USA Background/Aims: The HIV Tat protein is essential for amplifying viral genome transcription and represents a Figure (abstract: 2.6– 00158). Abstracts Journal of Virus Eradication 10S (2024) 7
promising antiviral target. Tat binds HIV mRNA’s stem-bulge loop structure TAR, to activate transcription. Block-and-lock approaches to cure HIV leverage transcriptional inhibitors to limit viral rebound upon treatment interruption by promoting long-lasting epigenetic silencing. The Tat inhibitor, dCA, was shown to promote long-lasting epigenetic suppression of the HIV promoter (LTR). The cost and difficulty of dCA synthesis have spurred the search for simpler inhibitors that possess exceptional drug-like properties while being more affordable to synthesize. Methods: The HeLa-CD4 cell-based Tat-transactivation assay of the HIV LTR driving luciferase was used for high throughput screening (HTS) of 579,443 small molecules, with dCA as a control. We followed up on hits with appropriate counter-screens and validations in physiologically relevant cells. Hit specificity was confirmed through various methods, including the use of Tat-defective viruses. Molecular docking and mutagenesis studies defined their binding site. The 26S proteasomal pathway engagement was identified using competitive inhibitors and gene depletions. Their efficacy was studied in front line therapy alongside ART, in the bone marrow-liver-thymus (BLT) mouse model of HIV latency and persistence. Results: We identified three compounds through HTS with therapeutic indices ranging from 5 to 181, exhibiting favorable chemical properties and cost-effective synthesis. They inhibited acute HIV replication in multiple cell models including human CD4+ T cells, without impacting cell viability. Moreover, these blocked viral reactivation in cell models of HIV latency, demonstrating their potential as block-and-lock agents. We showed that these compounds specifically promote the degradation of Tat across multiple systems, acting as molecular glues that enhance Tat ubiquitination and degradation via the 26S proteasomal pathway. Molecular docking and mutagenesis analysis revealed binding to the conserved residues Y26-F38R52. Improved analogs were also developed. In infected BLT mice treated with ART and Tat inhibitors, viral RNA levels in multiple organs and the size of the established reservoir were significantly reduced. Conclusion: We identified three novel small molecules that define a unique class of anti-HIV drugs that inhibit Tat via engagement of proteasomal degradation. Protein degradation may reduce viral resistance and block Tat’s pleiotropic activities. Any conflict of interests: I have no conflict of interests. Journal of Virus Eradication 10S (2024) 100405 https://10.1016/j.jve.2024.100405 3.2– 00080 New PKC Modulator Latency Reversing Agents for depleting persistent HIV reservoirs J.Moran1,T.Chou1, Z.O. Gentry2, O.D. Mcateer2, J.L. Hamad2, J.T.Kim3, P. A. Wender2, J.A. Zack3, M.D. Marsden1,* 1University of California - Irvine, United States; 2Stanford University - Stanford, United States; 3University of California - Los Angeles, United States Background/Aims: Latently infected cells are the major barrier preventing cure of HIV infection with antiretroviral therapy (ART) alone. Many promising strategies for HIV cure are currently being evaluated, and one of these involves inducing expression of latent virus with latency reversing agents (LRAs), allowing the host cell to be killed by viral cytopathic effects or immune effector mechanisms. For this strategy to be successful, safe and effective LRAs are needed to efficiently activate all latent virus in vivo. In the current study we evaluated different naturally occurring PKC modulators along with new designed, synthesized molecules intended to have improved HIV latency reversal and in vivo tolerability. These were tested in vitro and in vivo for their effects on HIV reservoirs. Figure (abstract: 3.1– 00145). Abstracts Journal of Virus Eradication 10S (2024) 8
Methods: A diverse collection of new PKC modulators based on different naturally occurring PKC scaffolds were designed and synthesized. These included slow-release prodrugs that are intended to improve in vivo tolerability through altered biodistribution of active compounds, and molecules with promising and selective in vitro PKC binding properties. These compounds were tested using in vitro HIV latency models, primary cells, and in vivo using HIV-infected humanized mice treated with ART. Results: Several compounds showed significantly improved latency reversal properties in vitro compared with both naturally occurring compounds and our best previously synthesized analogs. These compounds also synergized with other classes of LRA. Conversion of compounds to slow-release prodrugs resulted in a significant increase in the range of bioactive in vivo tolerated doses and thus expanded the potential “therapeutic window” for administration. In HIV infected ART-treated humanized mice, synthetic PKC modulators were capable of inducing expression of latent HIV, and delaying or preventing rebound if ART is stopped, indicating a reduction in rebound-competent viral reservoirs. Conclusion: This work demonstrates that several new LRAs that function through the PKC pathway are capable of potently reversing HIV from latency and reducing rebound-competent HIV reservoirs present during ART. They are also better-tolerated in vivo than the natural compounds on which they are based. Hence, this approach shows promise for use in HIV cure strategies. Any conflict of interests: Stanford University has filed patent applications on synthetic PKC modulators and related technology, which has been licensed by Neurotrope BioScience (Synaptogenix, Inc.) and Bryologyx Inc. P.A.W. is an adviser to both companies and a cofounder of the latter. J.A.Z. is a cofounder of CDR3 Therapeutics and is on the SAB of Bryologyx. The remaining authors declare no competing interests. Journal of Virus Eradication 10S (2024) 100406 https://10.1016/j.jve.2024.100406 3.3– 00093 RasGRP1 agonists induce cyclin T1 translation to reverse HIV-1 latency in primary CD4+ T cells U.Mbonye1,*, A. Bellomo2,3, E. Elhalem2,3, L. Gandolfi Donadio2,3, M. Julieta Comin2,3, J. Karn1 1Department of Molecular Biology & Microbiology, Case Western Reserve University School of Medicine - Cleveland, United States; 2Department of Active Ingredients and Biorefineries, National Institute of Industrial Technology - Buernos Aires, Argentina; 3National Scientific and Technical Research Council, Argentina Background/Aims: Reactivation of latent HIV-1 in memory CD4+ T cells requires the posttranscriptional biogenesis of P-TEFb, a transcription elongation factor whose cyclin T1 (CycT1) regulatory subunit is translationally repressed in the resting cellular state. Although it was long assumed that P-TEFb activation was mediated by a protein kinase C (PKC)-mediated pathway, we found that naturally occurring diacylglycerol (DAG)-mimicking PKC agonists stimulate CycT1 protein synthesis and reactivate latent HIV-1 primarily via the RasGRP1-Ras-Raf-MEK-ERK1/2 signaling pathway. Similarly, we’ve observed that synthetic agonists which preferentially bind RasGRP1 over PKC potently induce P-TEFb and reverse HIV-1 latency in primary CD4+ T cells. Therefore, targeting P-TEFb with synthetic DAG indololactones is a novel strategy for HIV latency reversal. Methods: Synthetic DAG indololactones, with known differential affinities for PKC and RasGRP1, were tested for their ability to stimulate P-TEFb expression in memory CD4+ T cells. Combinatorial latency reversal studies were performed in a primary T-cell latency model to examine the effectiveness of DAG indololactones at synergizing with LRAs that stimulate proviral transcription initiation. We also utilized these agents as experimental tools to define the signaling mechanisms by which RasGRP1-Ras-Raf-MEK-ERK1/2 can overcome the translational repression of CycT1 in memory T cells. Results: Three of the 4 DAG indololactones tested effectively stimulated CycT1 protein synthesis and generated active P-TEFb at concentrations below the threshold needed to elicit expression of the T-cell activation markers CD69 and CD25. The DAG indololactone 2A-127, which preferentially binds RasGRP1 over PKC-α more than 60-fold, synergized with the HDAC inhibitors SAHA, panobinostat, and romidepsin in reactivating latent HIV-1 in primary T cells. DAG-mimicking agonists were observed to specifically activate mRNA translation in memory T cells through a RasGRP1-ERK1/2-dependent mechanism that simultaneously stimulates p90 ribosomal S6 kinase and mTORC1 activities. Conclusion: DAG indololactones can stimulate the posttranscriptional expression of P-TEFb in memory T cells at concentrations below the threshold needed to induce T-cell activation, likely through a RasGRP1-mediated ERK1/2-mTORC1-S6K-rpS6 pathway. These agents can synergize with HDAC inhibitors, thereby bolstering the hypothesis that a two-pronged LRA strategy that targets P-TEFb biogenesis and enables RNA polymerase II recruitment to the HIV-1 promoter is needed to reverse HIV-1 latency efficiently. Any conflict of interests: No conflicts of interest to declare. Journal of Virus Eradication 10S (2024) 100407 https://10.1016/j.jve.2024.100407 3.4– 00150 Unbiased Genome-Wide CRISPR Screens in Primary Human CD4+ T Cells Identify Novel Proviral and Anti-viral HIV Host Factors U. Rathore1,*, E. Dugan1, N.J. Krogan1, A.Marson1,* 1Gladstone Institutes, University of California, San Francisco - San Francisco, United States This work is supported by NIH/NIAID funding for the HIV Accessory & Regulatory Complexes (HARC) Center grant P50 AI150476 (A.M., N.J.K.). Background/Aims: Human immunodeficiency virus (HIV) relies on host cellular machinery for replication, persistence, and dissemination. Identifying host factors that either promote or restrict HIV infection is crucial for developing effective therapies. However, the lack of unbiased, genome-scale CRISPR screening methods in physiologically relevant primary human CD4+ T cells has hindered our understanding of these critical interactions. Methods: We developed and executed the first-ever genomewide CRISPR knockout (CRISPRn) and CRISPR activation (CRISPRa) screens in primary human CD4+ T cells to systematically identify proviral and antiviral host factors involved in HIV infection. These screens were integrated with single-cell Abstracts Journal of Virus Eradication 10S (2024) 9
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