CROI 2024 Abstract eBook

Abstract eBook

Poster Abstracts

most detectable directly preceding rebound viremia, when the viral load has not reached the limit of detection and is still under tentative immune control. Methods: To investigate changes in the immune system indicative of the first signs of viral rebound, we performed two separate analyses on a cohort of donors with HIV-1 who participated in an observational analytical treatment interruption (ATI). We performed single cell RNAseq on peripheral blood mononuclear cells (PBMCs) from a subset of 10 donors and analyzed soluble protein levels in plasma using three Olink 92-plex panels on 19 donors. For the purposes of analysis, we compared three distinct timepoints: on-ART (before ATI), pre-rebound (during ATI, before detectable viremia), and post-rebound (detectable viremia). scRNAseq data was analyzed using the Seurat integration workflow. Pathway analysis using MSigDB hallmark gene sets collection was performed on scRNAseq data. Olink protein levels were log-normalized and assessed with a paired Wilcox test. Results: In these PLWH, we found a significant increase in monocyte subsets prior to detectable viral rebound. Non-classical inflammatory CD14-CD16++ monocytes increased in proportion by 1.9% (p=0.008) while classical CD14++CD16- monocytes increased in proportion by 3.6% (p=0.054) (Fig. 1). Pathway analysis indicated enrichment for IFNγ, IFNα, and inflammatory response pathways within monocyte subsets. Olink analysis indicated an increase in soluble inflammatory plasma proteins post-rebound related to immune activation (PD-L2, TRAIL), cytokine signaling (TNF, LAG3, GZMB), and NFkB signaling (CXCL9, CXCL10, IL12). Conclusion: Our data suggest that prior to detectable viremia, a subset of circulating monocytes activate and expand in response to low level viral replication. These cells shift to a more inflammatory phenotype, indicating innate immune sensing of early viral rebound activity. This has the potential to be a non-viral biomarker of imminent viral rebound. Intact Proviruses Persist in Expressed Genes in People With Non Suppressible HIV on Long-Term ART Joshua A Gluck 1 , Sean Patro 2 , Elias K. Halvas 3 , Kevin Joseph 3 , Nathan McKenna 3 , Shuang Guo 2 , Shadab Parvez 1 , Jason W. Rausch 1 , Xiaolin Wu 2 , John M. Coffin 4 , John W. Mellors 3 , Stephen Hughes 1 , Mary F. Kearney 1 1 National Cancer Institute, Frederick, MD, USA, 2 Leidos Biomedical Research, Inc, Frederick, MD, USA, 3 University of Pittsburgh, Pittsburgh, PA, USA, 4 Tufts University, Boston, MA, USA Background: The persistence of replication-competent proviruses during ART is a key barrier to an HIV cure. A previous study identified 3 cell clones carrying replication-competent proviruses that were integrated in genes and were the sources of non-suppressible viremia (NSV). Here, we asked if additional clones carrying intact proviruses were present in these 3 individuals; and, if so, where the proviruses were integrated in the host genome. Methods: Three donors on ART for 10-20 years who recently developed NSV were sampled over 1-4 years. PBMCs were subjected to endpoint diluted multiple displacement amplification (MDA). MDA wells were screened for HIV LTR, psi (Ψ), and RRE. Proviruses that were positive for LTR, Ψ, and RRE underwent full-length HIV sequencing and integration sites analysis. The % predicted-intact proviruses were calculated as in the IPDA and also using HIV LTR+ MDA wells as the denominator. A custom pipeline was used to map both discrete and non-discrete (e.g., centromeric) integration sites. Gene expression levels in memory CD4+ T cells were determined using the Human Protein Atlas Database (www.proteinatlas.org). Results: We identified 7 additional clones carrying sequence-intact HIV proviruses (10 total). All 10 integration sites mapped to expressed genes (Figure) and 4 were in the same orientation as the gene. Five were in KRAB-ZNF genes (50%), compared to the 3.7% of total proviruses (which are mostly defective) in KRAB-ZNF genes in one of the donors (p<10 -4 ). Reported gene expression levels were not significantly different for the genes with intact vs defective proviruses (median=33.9 vs 61.0 TPM; p=0.3). Predicted-intact proviruses (Ψ+RRE) comprised 9-10% of the total population using the IPDA approach and

comparing to HIV-negative individuals. However, notably, in individuals treated during chronic infection as compared to HIV negative individuals, TLR/RLR crosstalk was impaired: the level of IL12 and IL27 was 2 to 1.5 fold lower and, and IL6 restriction decreased from 1.9 to 1.2 fold. Conclusion: Crosstalk between TLR8 and RLR is disrupted following chronic infection with HIV-1, initiation of ART in the acute phase of infection preserves this crosstalk to a large extent. Our data suggest that HIV- related monocyte dysfunction might underlie this lack of crosstalk, as monocytes respond to this form of TLR8 and RLR stimulation. Differences between treated acute and chronic infection suggest that this phenomenon is more pronounced during chronically treated infection. Deciphering these alterations in crosstalk is paramount for pioneering strategies that bolster immunity and eradicate reservoirs, especially in a chronic setting. The Effect of HIV Infection at Pre-ART Initiation on the Early Innate Immune System Vinh B Dinh 1 , Lesley de Armas 1 , Rajendra Pahwa 1 , Suresh Pallikkuth 1 , Christine Dang 1 , Stefano Rinaldi 1 , Nicola Cotugno 2 , Paolo Palma 2 , Nadia Sitoe 3 , Paula Vaz 4 , Maria Grazia Lain 4 , Savita Pahwa 1 1 University of Miami, Miami, FL, USA, 2 Bambino Gesu Children's Hospital, Rome, Italy, 3 Instituto Nacional de Saúde, Maputo, Mozambique, 4 Fundação Ariel Glaser Contra o SIDA Pediátrico, Maputo, Mozambique Background: Despite the advent of antiretroviral therapy (ART), perinatal HIV infection still occurs, mostly in low-income countries. The nature of the innate immune response to HIV infection in infants is relatively unknown, as is an understanding of the effect of HIV replication on the development of the early innate immune system. Methods: 68 perinatally HIV exposed infants from Maputo, Mozambique were diagnosed as infected (HEI, n=33) not (HEU, n=35) at 1 month of age, prescribed ART for HEI and followed until 24 months. PBMC were isolated from blood at the clinical site and cryopreserved before being shipped to Miami. Flow cytometry was performed using a 27-color panel on PBMC to investigate the effect of HIV on innate immune cells (NK and monocytes). NK cell subsets were defined based on CD56 and CD16 expression on lineage negative lymphocytes. Monocytes were identified based on CD14 and CD16 expression on lineage negative and HLA- DR positive cells. These cell types were analyzed for markers of immune activation (IA), immune regulation, and trafficking. A flow cytometry based assay was implemented to investigate the killing potential and degranulation of NK cells utilizing a highly sensitive in vitro target (K562) and HIV-Infected target cell line (HUT78/SF2) in a small subset of the cohort. Results: At study entry pre-ART, NK cells in HEI infants exhibited an altered profile of activation, inhibitory, and trafficking receptors suggestive of a more activated profile when compared to HEU such as increased activation (CD38) and trafficking (CCR2) with decreased inhibition (NKG2A). In addition, similar alterations were observed in the monocyte compartment but to a lesser extent. CCR2 on NK cells and intermediate monocytes were highly correlated with pre-ART viral load. NK cells of HEI had a similar cytotoxic capacity as HEU towards both cell lines but increased degranulation towards the K562 cell line. By 10 months of age, disturbances in subset distribution and markers of activation, inhibition, and trafficking were partially reversed in both viremic and aviremic infants. Conclusion: The data suggests that NK cells and monocytes of HEI have an activated profile that appears to diminish with age. In addition, these cells are associated with viral replication at pre-ART initiation. These observations warrant further investigation on the impact of HIV on development of the innate immune system and the role of Intermediate monocytes and NK cells on viral replication and reservoir establishment. Inflammatory Monocytes Increase During HIV-1 Treatment Interruption Before Detectable Viremia Natalia de la Force 1 , Anna Farrell-Sherman 1 , Renan Valieris 2 , Steven G. Deeks 3 , Israel Tojal Da Silva 2 , Timothy J. Henrich 3 , Lillian Cohn 1 1 Fred Hutchinson Cancer Center, Seattle, WA, USA, 2 AC Camargo Cancer Center, São Paulo, Brazil, 3 University of California San Francisco, San Francisco, CA, USA Background: When antiretroviral therapy (ART) is interrupted, most people living with HIV (PLWH) experience rapid viral rebound within a few weeks. We currently have no reliable non-viral biomarkers of imminent viral rebound. Rebound viremia is defined by circulating HIV RNA, but we hypothesize that low levels of virus replicating in tissues may create measurable biological changes in the immune landscape prior to detectable viremia. These changes may be

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