CROI 2024 Abstract eBook

Abstract eBook

Poster Abstracts

Results: Post-purification sample characterization by imaging cytometry revealed a small (median of 0,015% of CD4+ T cells express CD32) and pure (>96%) CD32+CD4+ T-cell population. CD32 protein expression on these cells was evidenced by a dim but homogeneous signal, spread over the cellular membrane. To characterize this subset, we performed single-cell RNA sequencing and observed clustering in the memory T cell compartment with Th1, Th2, and cytotoxic gene signatures. These transcriptional traits were confirmed on protein level, as significant increases in Perforin, Granzyme A and K, as well as IFN-γ, IL4, and IL10 production were measured after short-term in vitro stimulation (all p-values <0.05). Detailed FACS phenotyping revealed that 80% of CD32+CD4+ T cells make part of the central memory, effector memory and transitional memory compartment the latter expressing high levels of CD31, a protein with a key role in leukocyte transmigration. Median fluorescence intensity on CD32+CD4+ cells was significantly increased for HLA-DR, PD-1 and CD95 (all p values <0.05). Importantly, in HIV-1 infected individuals on ART, these pure CD32+CD4+ T cells were on average 284-fold enriched for HIV-1 DNA. Conclusion: We found that CD32+CD4+ T cells are memory CD4+ T cells with traits of activation harboring cytotoxic gene and protein signatures. The tremendous enrichment in HIV-1 DNA suggests that these cellular traits represent an ideal niche for HIV-1 to persist during ART. Persistence of HIV-RNA in Autopsy Tissue Samples From Persons With HIV With Suppressed Viral Loads Hiromi Imamichi 1 , Ven Natarajan 2 , Francesca Scrimieri 2 , Mindy Smith 1 , Yunden Badralmaa 2 , Marjorie Bosche 2 , Jack Hensien 1 , Thomas Buerkert 1 , Weizhong Chang 2 , Brad Sherman 2 , Kanal Singh 1 , H. Clifford Lane 1 1 National Institute of Allergy and Infectious Diseases, Bethesda, MD, USA, 2 Frederick National Laboratory for Cancer Research, Frederick, MD, USA Background: The rapid viral rebound observed following treatment interruption, despite prolonged time on ART with plasma HIV-RNA levels <40 copies/ml, suggests persistent HIV-1 reservoirs outside of the blood. However, the relative contribution of different anatomical compartments to the HIV-1 reservoir is not well understood. Methods: Autopsy specimens were collected from 10 donors: 3 with active HIV-1 replication (blood HIV-RNA levels ≥5 copies/1 µg host-genomic RNA) ("Active") and 7 with suppressed HIV-1 replication (blood HIV-RNA levels <5 copies/1 µg host-genomic RNA)("Suppressed") at the time of death. Specimens were collected from 31 different tissues compartments including blood. Levels of HIV-DNA and HIV-RNA were determined using quantitative PCR. HIV-1 proviruses were analyzed by 5'LTR-to-3'LTR PCR single-genome amplification of near full-length HIV-1 (9 kb in size) and direct amplicon sequencing. A total of 1,902 HIV-1 sequences derived from the 10 donors (median: 128, range: 42 - 697 sequences per donor) were obtained and used for analyses. Results: HIV-DNA and HIV-RNA species were detected in all 10 donors in 31 different tissue compartments. No statistically significant differences were noted between the Active and Suppressed groups when comparing levels of HIV-DNA in blood or tissues. The same was true for levels of HIV-RNA species in tissues other than blood. The frequencies of full-length intact HIV-1 provirus (encoding replication-competent virus) were similar between the Active and Suppressed groups (5.9% vs. 3.7%, p=0.18). In both populations, the highest levels of HIV-RNA expression were within lymphoid tissues and the ileum. Multiple copies of identical full-length intact HIV-1 provirus were found in one donor in the Suppressed group. The expanded clone was detected in the blood, the kidney and the liver but not associated with HIV-RNA expression. Conclusion: Outside of peripheral blood, similar levels of HIV-DNA and HIV-RNA expression were noted in autopsy samples from persons with HIV with either active or suppressed HIV-1. These data imply that the substantial seeding of tissues with cells harboring proviral DNA seen in the setting of HIV-1 infection does not change despite ART and that many of these cells are actively transcribing HIV-RNA. While it is likely that the majority of this HIV-RNA is coming from cells harboring defective proviruses in the suppressed population, these findings highlight one of the challenges in achieving an HIV cure.

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Development of a Single-Cell Multiomic Assay to Phenotype SIV/SHIV Infected Rhesus Macaque Cells Jayme M Nordin , Vincent H. Wu, M. Betina Pampena, Michael R. Betts University of Pennsylvania, Philadelphia, PA, USA Background: SIV infection recapitulates many aspects of HIV-1 infection. However, the SIV reservoir at the single-cell resolution remains to be defined. Here, we adapted our single-cell viral Assay for Transposase Accessible Chromatin with Select Antigen Profiling by sequencing (viral ASAPseq) to rhesus macaque SIV infection model, to characterize SIV/SHIV reservoir epigenetics and phenotype. Methods: To adapt viral ASAPseq to the nonhuman primate model, we compiled, titrated, and characterized a rhesus cross-reactive antibody panel consisting of 62 immune-related markers using custom and commercially available oligo-tagged antibodies from Biolegend. Using this cocktail, we performed viral ASAPseq on purified lymph node CD4+ T cells at day 13 post-SIVmac251 infection. Finally, we developed a customized bioinformatics analysis pipeline to assess infected cells after viral alignments were made against the SIVmac251 sequence. Results: From this pilot test, we identified 81 SIV+ cells out of 15,334 cells (0.5%); of these, 42 cells had 2 or more reads aligned to multiple regions of the provirus. Using both the epigenetic and surface antigen profiles, we found that 67% of infected cells had characteristics of T follicular helper, T follicular regulatory, or T regulatory cells. The remaining infected cells appeared to have effector, activated, and/or resident memory characteristics. Conclusion: These initial findings demonstrate that NHP viral single-cell ASAPseq can be used to identify SIV+ cells and determine the unperturbed epigenetics and phenotype of these cells. Notably, this assay allows an unprecedented view into the SIV/SHIV reservoir to understand the perturbations associated with various cure strategies. Pure CD32+CD4+ Cells Are Cytotoxic Memory CD4+ T-Lymphocytes Highly Enriched for HIV-1 DNA Philipp Adams , Ben Berkhout, Alexander O. Pasternak Academic Medical Center, Amsterdam, Netherlands Background: Whether CD32 is a cellular marker of HIV-1 reservoir is a matter of ongoing debate. Despite intensive research, isolation of pure CD32+CD4+ T cells has not been reported thus far. Therefore, the biology of this cellular subset remains ill-defined. Here we present a novel purification strategy that allowed in-depth characterization of this subset in healthy donors as well as in HIV-1 infected individuals. Methods: A purification strategy was developed combining MACS pre purification of CD4+ T cells with subsequent double FACS sorting, validated by imaging cytometry. Single-cell RNA sequencing and in depth FACS phenotyping was performed followed by unsupervised analysis pipelines. Real time PCR was applied to quantify total HIV-1 DNA.

Poster Abstracts

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