# Novel ISH Approaches to Quantify Replication Competent Reservoirs

> **NIH NIH R01** · OREGON HEALTH & SCIENCE UNIVERSITY · 2020 · $705,511

## 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 individuals, 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 FOA
PA-17-305 "Imaging the Persistent HIV Reservoir" are to validate and apply novel microscopic and flow
cytometric RNA and DNA in situ hybridization (ISH) platforms that allow multiparametric single-cell
characterization of VR with various levels of residual transcription and/or translation. We will use these
approaches in HIV-infected samples and a NHP model of SIV infection, which allows detailed studies of diverse
tissues. In Aim 1, we will optimize our unique ISH platforms to identify and characterize ‘latent’ and ‘active’ VR
with putatively intact viral genomes and distinguish VR with different levels of transcriptional and translational
activities. We will define relationships between classical VR (e.g., PCR) and ISH-based measurements and
determine key differences between VR measures in the blood compared to different tissue environments, as
well as a comprehensive analysis of the “immune neighborhoods” and “inflammatory landscapes" in which VR
reside. In Aim 2, we will perform an interventional study using infection with SIVmac239M barcoded virus and
determine the relationship between novel ISH measures of VR in blood and tissues with time to viral rebound in
SIV-infected RMs after ART cess...

## Key facts

- **NIH application ID:** 9941040
- **Project number:** 5R01AI143411-02
- **Recipient organization:** OREGON HEALTH & SCIENCE UNIVERSITY
- **Principal Investigator:** JACOB D ESTES
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $705,511
- **Award type:** 5
- **Project period:** 2019-06-03 → 2024-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9941040

## Citation

> US National Institutes of Health, RePORTER application 9941040, Novel ISH Approaches to Quantify Replication Competent Reservoirs (5R01AI143411-02). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/9941040. Licensed CC0.

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