CROI 2025 Abstract eBook

Abstract eBook

Poster Abstracts

an HIV cure. Tuberculosis (TB) is the most prevalent opportunistic infection in people with HIV (PWH) but the impact of TB on the immune response to HIV, and the persistent HIV reservoir, is poorly understood. To address this issue, we studied the effect of TB/HIV coinfection on the persistence of cells infected with genetically-intact HIV. In addition, access to therapeutically aspirated pleural effusions (TB-PE) from persons living with HIV and TB allowed for a more in-depth analysis on the impact of the TB-microenvironment on HIV persistence and the functionality of anti-HIV CD8 T-cells. Methods: We used full-length individual proviral sequencing (FLIPS) to quantify the frequency of genetically-intact HIV in peripheral blood mononuclear cells (PBMCs) from 3 PWH and 3 individuals with TB/HIV. In one participant with TB/HIV coinfection, mononuclear cells from TB-PE (PEMCs) and PBMCs were also sequenced using FLIPS to characterize the site of the co-infection. To evaluate the impact of the TB microenvironment on HIV-specific cytotoxicity, CD8 T-cells from 4 PWH were cocultured with autologous HIV infected CD4 T-cells in the presence or absence of TB-PE. Moreover, the impact of TB-PE on the transcriptomic profile of CD8 T-cells was characterized by RNAseq in cells from 3 healthy donors. Results: Our analysis revealed a 3.2-fold increase in the frequency of cells infected with genetically-intact HIV in individuals with TB/HIV coinfection compared to PWH. Notably, within the individual with TB/HIV, PEMCs exhibited a 15.5-fold increase of intact HIV-infected cells compared to PBMCs. Interestingly, CD8 T-cells exposed to TB-PE demonstrated a significantly diminished capacity to eliminate HIV-infected cells (p=0.0021). Transcriptomic analysis showed that TB-PE downregulated pathways related to T-cell activation and the inflammatory response. Conclusions: These findings suggest that the TB-associated microenvironment hampers CD8 T-cell-mediated HIV control, promoting the persistence of genetically-intact HIV at the site of coinfection. This impaired cytotoxic response likely contributes to the persistence of the viral reservoir in the blood of individuals with TB/HIV coinfection. Our study underscores the significant challenge that TB poses to HIV curative strategies and highlights the need for targeted interventions for individuals living with HIV and TB. Species-Specific Splicing of DNA Editing Enzymes: Implications in HIV and SIV Mutagenesis Diako Ebrahimi 1 , Armando Mendez 2 , Rachael Rodriguez 1 , Kathryn A. Jackson Jones 3 , Margarita Rzhetskaya 3 , Nadeen Abdalla 2 , Luis Ferrández-Peral 4 , Fiona Carter-Tod 3 , Judd F. Hultquist 3 1 Texas Biomedical Research Institute, San Antonio, TX, USA, 2 University of Texas at San Antonio, San Antonio, TX, USA, 3 Northwestern University, Chicago, IL, USA, 4 University of Basel, Basel, Switzerland Background: Humans and nonhuman primates (NHPs) possess seven APOBEC3 (A3) enzymes, which restrict various viruses, notably HIV in humans and SIV in NHPs. These antiviral proteins inhibit viral infection by inducing G>A mutations in viral genomes and/or through mutation-independent mechanisms. HIV and SIV counteract A3 enzymes via their virion infectivity factor (Vif), which promotes A3 proteasomal degradation. Despite this, G>A mutations remain prevalent in viral genomes. A3G mainly induces GG>AG mutations, which frequently generate stop codons, while other A3 enzymes induce GA>AA changes, are less mutagenic, and do not produce new stop codons. Methods: We analyzed all HIV and SIV sequences available at the Los Alamos National Laboratory (LANL) databases and publicly available RNA-seq data from Sequence Read Archive (SRA). Sashimi plots were used to visualize mRNA splicing. Deaminase activity and dinucleotide substrate preferences were assessed via oligo-based deaminase assays. Oxford Nanopore Technologies (ONT) MinION Long-read sequencing incorporating adaptive sampling (AdS) was used to obtain full-length A3 isoforms. Results: Our analyses revealed species-specific G>A hypermutation and mRNA splicing patterns of A3 enzymes. Specifically, we found mRNA splicing defects in Old-World monkey A3Gs resulting in a significantly reduced level of GG>AG mutations in SIV compared to HIV. Furthermore, a 48-hour ONT MinION sequencing run on rhesus macaque PBMCs using AdS depletion achieved 8x-302x coverage of A3 enzymes despite their low expression. This analysis identified novel variants of A3A, A3C, A3G, and A3H, and revealed additional splicing defects in these genes and many other genes across human and NHP models. Conclusions: A3 and many other genes undergo species-specific splicing, highlighting the need for understanding the functional relevance of these molecular differences across animal models and humans to ensure that research

findings in animal models are translatable. We are currently conducting functional assays to assess the role of A3 splice variants in species-specific anti-viral immunity. Our long-term goal is to extend this study to other immune genes. Transcriptionally Active Defective HIV-1 Proviruses Persist in All CD4 T-Cell Subsets During ART Hiromi Imamichi 1 , Francesca Scrimieri 2 , Shweta Mistry 3 , Birgit Grund 3 , H. Clifford Lane 1 , Jason Baker 3 , for the INSIGHT START Study Group 1 National Institute of Allergy and Infectious Diseases, Baltimore, MD, USA, 2 Frederick National Laboratory for Cancer Research, Frederick, MD, USA, 3 University of Minnesota, Minneapolis, MN, USA Background: More than 95% of HIV-1 proviruses detected are defective but often associated with RNA transcripts during antiretroviral therapy (ART). These transcriptionally active defective proviruses may contribute to persistent immune activation seen in people with HIV (PWH) receiving ART. The distribution and persistence of these transcriptionally active defective proviruses has not been described. Methods: 11 PWH originally enrolled in the Strategic Timing of Antiretroviral Therapy (START) trial were studied. Levels of HIV-DNA and total HIV-RNA in frozen whole blood were determined at the baseline and 6-11 years later by real-time PCR. Also, at the 2nd visit, CD4 cell subsets were isolated by flow cytometry sorting with CD45RO and CD27 antibody staining to identify naïve (NA), central memory (CM) and effector memory (EM) subsets. Sequences of HIV-DNA and cell-associated (CA) HIV-RNA in CD4 subsets were characterized by 5'LTR-to-3'LTR PCR single-genome amplification and direct amplicon sequencing. Levels of HIV-DNA and CA HIV-RNA in the CD4 subsets were determined per-cell as well as per 1mL of blood. Results: All 11 participants (pts) were men, 55% white, 18% black, 9% Latino, median age 41 yrs, median baseline CD4 645 cells/ µ L. At the 2nd visit, after 4-11 yrs of suppressive ART, all 11 pts had ~2-log reduction in total HIV-RNA levels. While HIV-DNA levels also decreased, reductions were modest, at a median 0.5-log decrease. When analyzed on a per-cell basis, CA HIV-RNA levels were highest in EM, followed by CM and NA cells. In contrast, when analyzed based on total contribution per 1 mL of blood, CM cells had the highest median CA HIV-RNA levels, followed by EM and NA cells (Fig A). Of note, HIV-1 proviruses in the CD4 cell subsets were mostly defective, with median 9% of proviruses in a full-length intact form in NA cells, 4% in CM cells, and 2% in EM cells (Fig B). There was no evidence of transcription from these intact proviruses. Among 186 expanded provirus clones identified in 11 pts (2,473 seqs), 4% were shared among all CD4 cell subsets, with 43% of these being transcriptionally active. Conclusions: Transcriptionally active “defective” HIV-1 proviruses were present in all CD4 subsets in a cohort of 11 pts after 4-11 yrs of ART. The majority resided in CM cells, identifying the CM cell pool as an important source for long-term HIV-1 transcriptional persistence on ART. This has implications for the mechanism of persistent immune activation among ART-treated pts. Utilizing Defective HIV-1 Provirus as a Novel “Barcode” for Studying T-Cell Dynamics Vinie Kouamou 1 , Mindy Smith 1 , Brad T. Sherman 1 , Catherine Rehm 1 , Weizhong Chang 1 , H. Clifford Lane 2 , Hiromi Imamichi 2 1 National Institutes of Health, Bethesda, MD, USA, 2 National Institute of Allergy and Infectious Diseases, Baltimore, MD, USA Background: Achieving an understanding of T-cell dynamics and distribution of proviral DNA within T cell subsets is of critical importance in achieving a better understanding of the relationships between effector memory, central memory and naïve CD4 cell subsets. In this study, we investigated the relationships between these subsets utilizing the natural “barcoding” provided by replication-incompetent defective HIV-1 proviruses. The provirus-barcode gives each single cell a unique identity, which is passed on to daughter cells without the signal being diluted during somatic cell division. Methods: Cryopreserved cells were obtained from 2013 to 2023 from a 54 year-old white male with HIV-1 infection. During this time, his plasma HIV-RNA levels remained <20-40 copies/mL with transient viral blips below 454 copies/ mL, and his median CD4 count was 651 cells/µL (range: 434-756 cells/µL). CD4 cell subsets were isolated by flow cytometry sorting with a combination of CD45RO and CD27 antibody staining to identify naïve (NA, CD27+CD45RO-), central memory (CM, CD27+CD45RO+) and effector memory (EM, CD27- The figure, table, or graphic for this abstract has been removed.

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CROI 2025