# Viral ASAPseq definition of CD4+ T cell viral reservoirs

> **NIH NIH R21** · UNIVERSITY OF PENNSYLVANIA · 2022 · $243,750

## Abstract

While combination antiretroviral therapy (ART) effectively controls HIV viremia in most people living with HIV
(PLWH), broadly applicable strategies to induce viral remission have remained elusive due to HIV reservoir
persistence. Substantial progress has been made in defining the virological characteristics of the proviral
reservoir of latently infected cells during ART. However, significant gaps remain in our understanding of the
infected CD4+ T cells that constitute the cellular HIV reservoir that limit our ability to develop effective
strategies for HIV cure. We have developed a novel high throughput 10X Genomics single cell assay to directly
identify HIV infected cells via the presence of integrated proviral DNA. Termed "viral ASAPseq" [Assay for
Transposase Accessible Chomatin (ATAC) Surface Antigen Profile sequencing, ASAPseq, with viral
alignment, viral ASAPseq], this assay allows detection of HIV proviral DNA within euchromatin in combination
with 154-marker cell surface antigen labeling, yielding multimodal single cell resolution of HIV infected cells.
This assay does not require ex vivo activation to detect the presence of integrated viral DNA, thereby
preserving the native state of the infected cell. We have successfully piloted this procedure in several
conditions, including in vitro infected primary CD4+ T cells, primary lymph node CD4+ T cells from viremic
PLWH, and peripheral blood CD4+ T cells from ART treated PLWH. We have further developed bioinformatic
strategies to identify proviral DNA using autologous viral sequence alignment, define the surface marker
composition of HIV infected cells, and characterize the epigenetic structure of infected cells. Thus, we are
poised for the first time to gain a direct understanding of HIV reservoir composition directly ex vivo. Here we
will apply this new technique to test the hypothesis that HIV infected CD4+ T cells have a cell surface or
epigenetic signature distinct from uninfected CD4+ T cells in ART treated PLWH. We will further pilot a new
strategy, scGETseq, capable of simultaneously and separately sequencing euchromatin and heterochromatin
to distinguish and profile integrated HIV provirus within the active and latent reservoir of ART treated PLWH.
Together our studies have the potential to provide the first full understanding of the unmanipulated CD4+ T cell
HIV reservoir directly ex vivo.

## Key facts

- **NIH application ID:** 10548385
- **Project number:** 1R21AI172629-01
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** Michael R Betts
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $243,750
- **Award type:** 1
- **Project period:** 2022-06-03 → 2024-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10548385

## Citation

> US National Institutes of Health, RePORTER application 10548385, Viral ASAPseq definition of CD4+ T cell viral reservoirs (1R21AI172629-01). Retrieved via AI Analytics 2026-05-28 from https://api.ai-analytics.org/grant/nih/10548385. Licensed CC0.

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