# Defining Mechanisms of Viral Persistence in Situ at the Single-Cell Level

> **NIH NIH R01** · OREGON HEALTH & SCIENCE UNIVERSITY · 2021 · $800,128

## Abstract

PROJECT SUMMARY
The major obstacle to an 'HIV cure' is the persistence of viral reservoirs (VR) harboring replication competent
viral genomes that have the capacity to produce infectious virus. These VR persist for long periods of time, and
even after years of suppressive ART, the systemic spread of virus resumes within a few weeks upon cessation
of ART in all but exceptional cases. Effective cure strategies will need to dramatically reduce or eliminate VR
through safe and scalable approaches. It is currently thought that the major VR are long-lived latently infected
resting memory CD4+ T cells, which remain quiescent until they are stimulated by external cues to produce virus.
In addition to the truly latent VR, emerging data shows that in individuals on suppressive ART a subset of VR
transcribe viral RNA (vRNA+) at variable levels (termed ‘active VR’). In some cases, this might lead to residual
levels of HIV replication, particularly in tissue microenvironments where drug concentrations are suboptimal.
Even without full viral replication, this residual expression of virus may have adverse consequences and
contribute to chronic immune activation/inflammation and non-AIDS defining clinical events. Eradicating HIV will
require targeting both the ‘latent’ and ‘active’ VR, however, our current understanding of HIV reservoirs comes
mostly from studies performed in peripheral blood, but the blood contains only a small fraction of VR during ART.
We reason that to maximize efficacy of ‘HIV cure’ strategies, we need to first better characterize both the tissue
compartments and the cellular subsets from which infection might rebound in HIV-infected individuals after ART
is interrupted. Thus, the overarching goals of this research proposal, in response to RFA-AI-18-053 “Single-Cell
Multi-Omics of HIV Persistence”, is to merge our innovative in situ hybridization (ISH) approaches to quantify
and map VR at high resolution with multiple new cutting-edge multi-omics platforms to investigate mechanisms
of VR persistence at the single-cell level while retaining critically important contextual insight into the cellular
immune neighborhoods and inflammatory landscapes in which VR reside. In Aim 1, we will utilize our suite of
novel next-generation ISH (RNAscope, DNAscope and BASEscope) platforms to quantify and generate “atlases”
of ‘latent’ and ‘active’ VR longitudinally within tissue compartments (peripheral and mesenteric lymph nodes,
spleen, GI tract) before and at different timepoints during ART ± anti-inflammatory adjunctive therapy. In Aim 2,
we will perform an in-depth phenotypic analysis of VR and the cellular immune neighborhoods and inflammatory
landscapes in which they reside within tissues (guided by our high-resolution in situ VR mapping outlined above)
using Multiplexed Ion Beam Imaging (MIBI) proteomic analysis as well as unbiased SNaPP and nanoPOTs mass
spectrometry approaches for spatiotemporal molecular analyses on samples obtained by LCM of imm...

## Key facts

- **NIH application ID:** 10064126
- **Project number:** 5R01AI149672-02
- **Recipient organization:** OREGON HEALTH & SCIENCE UNIVERSITY
- **Principal Investigator:** JACOB D ESTES
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $800,128
- **Award type:** 5
- **Project period:** 2019-12-03 → 2024-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10064126

## Citation

> US National Institutes of Health, RePORTER application 10064126, Defining Mechanisms of Viral Persistence in Situ at the Single-Cell Level (5R01AI149672-02). Retrieved via AI Analytics 2026-06-11 from https://api.ai-analytics.org/grant/nih/10064126. Licensed CC0.

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