HIV-1 Cell-Associated RNA Assay

Cell-Associated RNA Assay Services for HIV-1 Research

The most direct measure of latency reversal available.

HIV cure researchers need a readout of viral transcription sensitive enough to detect induction of latent HIV-1 gene expression and specific enough that background proviral DNA is not counted as bona fide viral RNA.

Overview

While proviral DNA assays report how many infected cells are present, cell-associated HIV-1 RNA (CA-RNA) reports whether the proviruses in those cells are transcriptionally active, which is the most direct measure of latency reversal available.

Accelevir Diagnostics, a CAP-accredited, CLIA-certified bioanalytical laboratory in Baltimore, Maryland, provides the HIV-1 CA-RNA dPCR (CAR-dPCR) assay, a semi-automated duplex RT-dPCR assay that independently quantifies unspliced and total polyadenylated HIV-1 CA-RNA, performed alongside our proviral DNA, plasma viremia, and sequencing services in a single laboratory under one quality system, with qualification documentation available to support regulatory filings.

Request a technical consultation to scope your HIV-1 persistence measurement program.

One Laboratory, One Quality System

CA-RNA, intact proviral DNA, ultrasensitive plasma viremia, and near-full-length sequencing scoped against a single participant collection.

Baltimore, Maryland · BSL-2+ facility

Why Leading HIV Programs Trust Accelevir

Built for HIV Persistence Measurement

CAP/CLIA-Certified Laboratory

CAP-accredited, CLIA-certified high-complexity laboratory operating from a BSL-2+ facility in Baltimore, MD.

Rooted in HIV Cure Research

Rooted in the HIV-1 cure research community, with extensive experience in translational virology for latent and viremic patient cohorts.

Analytically Qualified Assays

Analytically qualified assays performed under a unified quality management system with qualification documentation available to support regulatory filings.

Trial-Ready Persistence Measurement

Assay services supporting HIV-1 persistence measurement across interventional cure and remission trials.

CA-RNA reports whether persistent proviruses are transcriptionally active.

What Sets Our HIV-1 CA-RNA Services Apart

Designed to Address the Limitations of Single-Plex RT‑qPCR

Most quantitative assays for HIV-1 CA-RNA employ single-plex RT‑qPCR, with quantitative precision and accuracy dependent on standard curves generated from contrived RNA or DNA controls.

These assays often vary considerably between performing laboratories, have generally not been analytically qualified, are frequently deployed without RNA-based controls, and are optimized only for subtype B HIV-1. Accelevir’s CAR-dPCR assay was designed to address each of these limitations.

Four Design Objectives

Subtype Coverage, Dual Targets, Specificity, and Control

Targets selected across Group M subtypes and major circulating recombinant forms.

01

Broad HIV-1 Subtype Coverage

Globally, most people infected with HIV-1 are infected with a non-Subtype B virus. However, most research assays for CA-RNA were developed and optimized for Subtype B, which limits their utility to global HIV-1 persistence research and cure trials. Primer and hydrolysis probe targets for our assay were selected following a comprehensive in silico analysis of publicly available HIV-1 sequences from each Group M subtype and major circulating recombinant form. We have demonstrated detection across subtypes using samples from PWH on ART with confirmed non-B subtype virus, as well as well-characterized isolates from a viral diversity panel. This means the assay can be used on any study population, without prior knowledge of the subtype of infection.

Both RNA targets are measured from the same prepared sample.

02

Two Independent Targets Quantified from a Single Sample

The assay independently quantifies unspliced HIV-1 RNA via a target in pol, and total polyadenylated HIV-1 RNA via a target spanning the polyadenylation signal in the 3′ LTR. Unspliced RNA is the transcript class with the largest body of comparative cohort data behind it, so your results can be read against the published literature rather than standing alone. Total polyadenylated RNA captures all correctly terminated spliced and unspliced transcripts, offering a comprehensive measurement of transcriptionally active proviruses. Measuring two independent targets also protects your dataset—a polymorphism at one primer or probe binding site doesn’t mean a complete non-detect, meaning you can retain more data on precious trial samples.

DNA contamination is eliminated at two stages of the RNA isolation workflow.

03

Specificity for Bona Fide Viral Transcripts

DNA contamination can bias RT-PCR results. In the case of HIV-1, this means provirus contamination and the artificial inflation of the number of unspliced HIV-1 transcripts. Accelevir RNA isolation protocols are optimized with this in mind, robustly eliminating DNA contamination at two stages of the workflow. Additionally, because the total polyadenylated target requires the poly(A) sequence, proviral DNA and commonly used HIV-1 plasmids are not detected. This means you can be confident that your data truly reflects CA-RNA load, free from proviral DNA contamination.

RT efficiency calibrators and per-sample spike-in controls travel with every batch.

04

Quality and Calibrator Controls at Every Step of the Workflow

Digital PCR provides absolute quantification of cDNA without dependence on an interpolated standard curve, but reverse transcribing RNA into cDNA is a sensitive process and its efficiency is never 100%. RT efficiency is an often-uncaptured component of RT-PCR assay accuracy, which can artificially reduce copies detected if not factored into downstream analysis. Our assay addresses this by including CA-RNA RT efficiency calibrators with every batch of cDNA, so we can capture any signal lost during cDNA synthesis. The calibrator is lot-controlled, copy number verified RNA template containing both CAR-dPCR assay targets, allowing for independent calibration of RT efficiency for each assay target. We also include an internal spike-in control for every sample prior to RNA isolation that must be detected for that sample to pass, confirming successful nucleic acid recovery on a per-sample basis rather than on a per-batch assumption. That means that every data point represents a correctly isolated, loaded, and analyzed sample, that can be accurately compared across runs and datasets.

Our HIV-1 CA-RNA Assay Services

CAR-dPCR for Quantifying HIV-1 Transcriptional Activity

HIV cure programs rarely rely on a single measurement. The HIV-1 latent reservoir is defined by multiple metrics—the genomic fidelity of integrated proviruses, and the capacity to transcribe HIV-1 RNA and produce infectious virions. With CAR-dPCR and our suite of HIV-1 proviral DNA assays, Accelevir provides HIV-1 assay services that can comprehensively measure every element of HIV-1 persistence—even on the same sample.

The transcriptional state of persistent proviruses is largely independent of their genomic integrity. While a fraction of proviruses may be permanently silenced or carry catastrophic defects that preclude transcription, the majority of both intact and defective proviruses appear capable of producing at least some viral transcripts. In people with HIV (PWH) on suppressive antiretroviral therapy (ART), CA-RNA is readily detectable at baseline, and a variety of viral RNA splice variants have been observed.

CAR-dPCR quantifies how much viral RNA infected cells are producing, making it the most definitive measurement when your endpoint is induction or suppression of HIV-1 gene expression rather than a change in the number of infected cells. DNA-free total cellular RNA is isolated from PBMCs and analyzed by duplex RT-dPCR, with unspliced and total polyadenylated HIV-1 RNA reported independently, a per-sample MS2 extraction control, and an RNA RT efficiency calibrator for quantification.

Duplex RT-dPCR quantifies both RNA targets from a single prepared sample.

CA-RNA, IPDA®, and Ultrasensitive Plasma Viral Load in Concert

Why Programs Combine These Measurements

The most common configuration in cure research pairs CAR-dPCR with the HIV-1 IPDA® and our ultrasensitive plasma viral load assay, and the three fit together because each resolves a different property of the same reservoir.

IPDA quantifies the frequency of cells carrying genomically intact, potentially rebound-competent proviruses, giving you the size of the persistent HIV-1 provirus population that matters. CA-RNA reports how transcriptionally active that population is. Ultrasensitive plasma viral load reports how much virus is reaching circulation in vivo. Together they answer a question none of them answers alone: whether an intervention reduced the reservoir, quieted it, or changed what it releases. That distinction is central to interpreting latency reversal, transcriptional silencing, and any therapy intended to reduce the infected cell population, and it is frequently what separates an interpretable result from an ambiguous one.

All three assays can be run from the same participant sample collection at Accelevir, under the same quality system, and an integrated report. For programs that also need reservoir composition or replication competence, near-full-length proviral sequencing and viral outgrowth are available from the same laboratory and can be scoped into the same study.

Applications

Where CA-RNA is the deciding endpoint

From latency reversal agent development to block-and-lock approaches, participant stratification, and cohorts in which the reservoir is very small.

Applications

Study Designs That Depend on a Transcriptional Readout

01 Latency Reversal Agent Development

Induction of CA-RNA is the standard pharmacodynamic endpoint for latency reversal, used to compare candidate agents and combinations in clinical trials to screen for potential responders and to monitor latency reversal during dosing. CA-RNA has been the readout in nearly every clinical trial of a candidate latency reversing agent to date, which puts your CAR-dPCR data in the context of the largest body of latency reversal research data available.

02 Transcriptional Silencing and Block-and-Lock Approaches

Approaches intended to durably suppress viral transcription rather than deplete the reservoir require a sensitive transcriptional readout, and in particular one that will not be confounded by background proviral DNA or readthrough transcripts, since these studies are seeking a decrease against an already low baseline. CAR-dPCR supports these interventions too, offering an ultra-sensitive readout of reduction in HIV-1 transcriptional activity in response to block-and-lock approaches.

03 Participant Screening and Stratification

Baseline CA-RNA levels correlate with the magnitude of latency reversal after treatment, which supports the utility of identifying likely responders prior to dosing. Pre-interruption transcriptional activity has also been associated with rebound timing across several cohorts, and CA-RNA is therefore commonly included in pre-interruption assessment alongside residual viremia and intact proviral DNA.

04 Cohorts in Which the Reservoir Is Very Small

In early-treated adults, pediatric cohorts, and individuals on very long-term ART, measures of reservoir activity can be particularly informative, and CA-RNA requires substantially less input material than culture-based methods. In pediatric studies, measuring CA-RNA offers a feasible alternative that can handle available blood volume constraints and still capture transcriptional activity of the reservoir.

05 Preclinical Studies

We also offer a variant of this assay to quantify viral CA-RNA from low input PBMC numbers across commonly used SIV and SHIV strains for preclinical rhesus macaque studies. Contact us to confirm current availability for your model.

06 Talk to a Scientist

Scope your HIV-1 persistence endpoints with our team.

FAQ

Frequently Asked Questions

Because these assays start with the same whole blood collection, they can be scoped against a single participant draw.

What is cell-associated HIV-1 RNA?

Cell-associated HIV-1 RNA is viral RNA present within infected cells, as distinct from cell-free virus circulating in plasma. It reflects the transcriptional activity of persistent proviruses. HIV-1 produces dozens of differentially spliced transcripts, and available assays differ substantially in which of these they detect.

How does CA-RNA differ from plasma viral load?

Plasma viral load measures virions that have been produced and released into circulation. CA-RNA measures transcription within cells, which occurs upstream of virion assembly and release. HIV-1 gene expression is subject to multiple post-transcriptional blocks, so transcription can occur without productive virion release, and a null plasma result does not preclude a transcriptional effect.

Why does polyadenylation matter in the assay design?

Requiring the poly(A) sequence excludes sources of signal that do not represent bona fide HIV-1 gene expression, such as residual proviral DNA and HIV-1 plasmids, which lack poly(A) entirely. Detecting DNA would bias a result toward an apparent treatment effect.

Does the assay perform across HIV-1 subtypes?

Yes, and this was a design objective rather than an afterthought. Primer and probe targets were selected for sequence conservation across Group M subtypes and major circulating recombinant forms following in silico analysis of publicly available sequences, and cross-subtype detection has been demonstrated using samples from PWH with confirmed non-B subtype virus and characterized isolates from a viral diversity panel. Studies enrolling outside North America and western Europe do not need to be designed around the limitations of a subtype B assay.

How is assay performance controlled?

Two controls, addressing two different quality metrics. An MS2 bacteriophage particle is spiked into every sample before extraction and must be detected for that sample to pass, which confirms successful isolation of RNA and loading of sample into the assay. A lot-controlled, copy number verified in vitro transcribed RNA standard containing both assay targets serves as the RT calibrator, normalizing results for RT efficiency for each target, as well as confirming RT performance within the expected range.

How do you handle precious PBMC samples from clinical studies?

The assay requires greater than 10 million viably cryopreserved PBMCs, or 10 million snap-frozen PBMC pellets. Accelevir’s PBMC processing services include validated thaw protocols, viability qualification, and quantification of CD4+ T cell frequency before RNA is isolated from cells. All samples remain under documented chain of custody with temperature-monitored storage. Input requirements differ across our menu, so groups running several assays on the same participants should plan collection volumes accordingly.

What regulatory standards does your CA-RNA assay meet?

Accelevir operates a CAP-accredited, CLIA-certified, high-complexity laboratory. The CAR-dPCR is an analytically qualified research-use only assay, performed under a unified quality management system, with a performance report provided to groups conducting clinical trials on request.

Which measurement should my study use?

CA-RNA quantifies how much viral RNA infected cells are producing and is the direct readout for interventions acting on viral gene expression. The HIV-1 IPDA® quantifies how many cells carry genomically intact proviruses. Ultrasensitive plasma viral load quantifies virion production and release. Many programs run all three, and because these assays are all performed on samples that start with whole blood collection and are performed in the same facility, they can be scoped against a single collection from a trial participant.

Start Your HIV-1 Persistence Measurement Project

Scope Your HIV-1 Persistence Measurement Program

Accelevir’s comprehensive HIV-1 persistence assay menu delivers the subtype coverage, process control, and qualification documentation that HIV cure programs require. Whether your study needs a single CA-RNA endpoint or an integrated profile combining CA-RNA, intact proviral DNA, and ultrasensitive plasma viral load, our Baltimore-based team is ready to scope your project.

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Technical consultation

Accelevir Diagnostics · Baltimore, MD · CAP/CLIA-certified laboratory