# Phenotypic and mechanistic analysis of the in vivo HIV latent reservoir by single-cell technologies

> **NIH NIH R01** · J. DAVID GLADSTONE INSTITUTES · 2022 · $843,541

## Abstract

PROJECT SUMMARY
 Combination antiretroviral therapy (ART) can suppress HIV replication and lead to decreased mortality
in HIV-infected individuals. However, ART does not eliminate the latent HIV reservoir, so effective viral
suppression requires lifelong ART administration. Therefore, developing a way to eliminate or achieve ART-free
control of the reservoir is a top research priority. One challenge to accomplishing this is that we still have limited
understanding of the phenotypic and functional properties of the latently-infected cells that persist in people living
with HIV. One challenge to characterizing in vivo latently-infected cells is the inability to directly phenotype these
cells, due to the lack of a universal biomarker distinguishing them from uninfected cells. As a result, the only way
to directly phenotype latent cells has been to stimulate a bulk population of patient-derived cells ex vivo in order
to induce expression of viral proteins by the latent cells. Although this allows identification and therefore
phenotyping of the reactivated cells by FACS, the measured phenotypes are different from the original
phenotypes of the latently-infected cells since ex vivo stimulation alters gene expression. Here, by applying a
pseudotime-based bioinformatics approach called PP-SLIDE on paired sets of unstimulated and stimulated
patient cells deep-phenotyped by high-dimensional single-cell analytical approaches (CyTOF, single-cell
RNAseq), we infer the phenotypes of latently-infected cells in their original pre-stimulation state, and use this
approach to chart the in vivo latent reservoir. In Aim 1, we will use PP-SLIDE on CyTOF-phenotyped cells to
compare the latently-infected cells present in the blood and tissues of clinically-matched men and women. In
Aim 2, we will characterize the extent to which markers identified in Aim 1, as well as markers identified from an
unbiased approach implementing PP-SLIDE on cells analyzed by single-cell RNAseq, enrich for reservoir cells
harboring HIV with genetically-intact replication-competent HIV, for these are the cells that are likely the most
important to control or eliminate for ART-free viral control. In Aim 3, we will characterize the mechanisms that
allow the latently-infected cells to persist, focusing on the role of antigen- and homeostasis-driven clonal
expansion of CD4+ T cells. By combining cutting-edge single-cell analysis tools with high-dimensional data
analysis methods to map the “atlas” of in vivo latent cells, our studies will provide an unprecedented definition of
the features of reservoir cells that persist, reveal whether any reservoir cell traits associate with anatomy (blood
vs. tissues) or biological sex (men vs. women), and inform on the mechanisms driving reservoir maintenance.
This knowledge that will be important for designing targeted methods to achieve a universal HIV cure.

## Key facts

- **NIH application ID:** 10448398
- **Project number:** 5R01AI147777-05
- **Recipient organization:** J. DAVID GLADSTONE INSTITUTES
- **Principal Investigator:** Nadia R Roan
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $843,541
- **Award type:** 5
- **Project period:** 2019-08-01 → 2025-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10448398

## Citation

> US National Institutes of Health, RePORTER application 10448398, Phenotypic and mechanistic analysis of the in vivo HIV latent reservoir by single-cell technologies (5R01AI147777-05). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10448398. Licensed CC0.

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