Poster Presentation Lorne Infection and Immunity 2023

Single-cell RNAseq analysis of HIV reservoir cells from viremic and virally-suppressed individuals living with HIV (#201)

Sushama Telwatte 1 2 3 , Julie Frouard 4 , Xiaoyu Luo 4 , Holly A Martin 3 , Nikhila G Kadiyala 3 , Adam Wedrychowski 3 , Pavitra Roychoudhury 5 6 , Rebecca Hoh 7 , Steven G Deeks 7 , Sulggi Lee 7 , Nadia Roan 4 , Steven A Yukl 2 3
  1. The Peter Doherty Institute for Infection and Immunity, Melbourne, VICTORIA, Australia
  2. Infectious Diseases, University of California, San Francisco, San Francisco, California, United States
  3. San Francisco VA Medical Center, San Francisco, California, United States
  4. Gladstone Institute of Virology and Immunology, University of California, San Francisco, San Francisco, California, United States
  5. Laboratory Medicine and Pathology, University of Washington, Seattle, Washington, United States
  6. Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States
  7. University of California, San Francisco, San Francisco, California, United States

Latently-infected CD4+ T cells are considered the main barrier to a cure for HIV-1. While these cells do not produce HIV constitutively, they can be induced to produce infectious virus upon activation. During viral suppression under therapy, a proportion of infected cells remain transcriptionally-active and these are predictive of time to viral rebound after antiretroviral therapy (ART) cessation. Considerable technical challenges are posed by the low frequency of transcriptionally-active HIV-1 reservoir cells and the fact that many of those cells reside in lymphoid tissues such as the gut. Finally, there are currently no known biomarkers that reliably distinguish latently-infected cells from uninfected cell populations in vivo.

To address these limitations, we developed “HIV-Seq”, a new single-cell (sc)RNAseq approach that enables simultaneous characterization of the transcriptome and surface proteome of unstimulated HIV-infected cells from blood and gut tissue from people living with HIV (PWH). Using custom-designed HIV-specific capture sequences and DNA-barcoded antibodies directed to key cell surface proteins (CITE-seq) introduced into a single cell RNAseq workflow (10X Genomics), we describe an in-depth combined scRNAseq/CITE-seq analysis of HIV reservoir cells from blood in the context of both viremia and ART suppression.

This new approach was applied to longitudinal samples collected at Week 0 (prior to commencing ART) of acute infection, and Week 24 or 45 after ART suppression [n=5]. CD4+ T cells were enriched using bead-based isolation and stained with DNA-tagged antibodies. HIV capture sequences were incorporated during library preparation. We additionally characterised total immune cells (CD45+) and T cells (CD3+) from the blood and gut of one ART-suppressed individual. scRNAseq and CITE-seq analyses were performed and sequences were aligned to a constructed subtype B consensus reference sequence.

Based on viremic sample data, HIV-seq enables 32-72% increased capture of HIV RNA+ cells, relative to no capture. In total, we identified 1232 HIV RNA+ cells from viremic timepoints and 26 HIV RNA+ cells from the ART-suppressed timepoints representing the transcriptionally-active reservoir.

Our HIV-seq method enables efficient identification and characterization of HIV-infected cells including in the context of ART suppression, allowing for in-depth transcriptomic and surface phenotypic analysis of transcriptionally-active reservoir cells.