HIV-seq Finds More Than 1,000 Active Reservoir Cells Standard Methods Miss

More than 38 million people worldwide live with HIV on antiretroviral therapy. The drugs work, suppressing the virus to undetectable levels in the blood. But the infected cells do not disappear. They persist for decades in what researchers call the HIV reservoir - a population of immune cells that harbor the virus in a state that evades both the drugs and the immune system. Understanding what those cells are actually doing, genetically, has been a stubborn technical problem. A new tool developed at Gladstone Institutes may have cracked it open.

The Gap That Standard Sequencing Left Open

In recent years, single-cell RNA sequencing has transformed biomedical research by allowing scientists to see which genes are active in individual cells. But when applied to blood samples from people with HIV on therapy, it consistently fell short: typically detecting only one or two HIV-infected cells per patient. That is far too few for meaningful statistical analysis.

Julie Frouard, PhD, a scientist in Nadia Roan lab at Gladstone and a first author of the study, identified the root cause. "When single-cell RNA sequencing was applied to blood samples from patients on therapy, it oftentimes only detected one or two of these cells per person. That is not enough for a meaningful analysis." The problem is technical: standard sequencing captures RNA fragments based on criteria that HIV RNA does not always meet. Infected cells that are still producing HIV RNA fragments get missed.

The solution was to build a new tool, purpose-designed for the virus. HIV-seq is a single-cell RNA analysis method that is specifically tailored to recognize cells producing HIV RNA. The work was done in collaboration with a team at the San Francisco Veterans Affairs Medical Center, led by physician-scientist Steven Yukl, MD.

Recovering the Cells That Were Slipping Through

The performance difference was decisive. When HIV-seq was run head-to-head with the standard approach, it recovered more HIV-infected cells and found higher numbers of HIV RNA within those cells. From people with active HIV infection who had not yet started therapy, HIV-seq recovered more than 1,000 reservoir cells from four patients - described by the team as the highest number to date from that kind of sample. From people on therapy, where reservoir cells are rare, the tool recovered 25 cells from just three patients.

"Now, for the first time, we can actually characterize these cells in a meaningful manner for people whose HIV is suppressed by antiretroviral therapy," said Yukl, also a professor of medicine at UCSF. The study was published March 3, 2026 in Nature Communications, with Roan as senior author.

Fiery Before Therapy, Quiet After

With enough cells to analyze, the team could directly compare HIV-infected cells from people before and after starting antiretroviral therapy. The differences were stark.

Cells from untreated patients had what the researchers described as cytotoxic features - proteins associated with the ability to kill other cells. They also had lower levels of specific genes linked to HIV suppression, suggesting the virus may actively inhibit those genes to replicate faster. "In a general sense, I would say that these cells were rather inflammatory, or fiery," said Roan, who is also a professor at UCSF.

In contrast, reservoir cells from patients on therapy were quieter. They lacked cytotoxic features, showed anti-inflammatory characteristics, and had higher levels of genes that help cells evade death and survive long-term. Those survival signals are potentially the answer to one of the most clinically important questions in HIV research: how do these cells survive for decades when the immune system should be eliminating them?

A Lead for Existing Clinical Trials

One of the proteins elevated in cells from treated patients is associated with a cellular survival pathway currently being targeted in an ongoing clinical trial. "Our data provide further support for that research," said Yukl. The HIV-seq findings may not only explain the reservoir biology but actively inform which therapeutic targets are worth pursuing.

Roan lab is already building on the results by testing, in laboratory models, whether reservoir cells can be stopped from multiplying by targeting these pro-survival pathways. The tool itself is the durable contribution - a more sensitive way to find and characterize the cells that make HIV so difficult to cure.

The research was supported by the National Institutes of Health, the California HIV/AIDS Research Program, UCSF-Bay Area CFAR, and the James B. Pendleton Foundation.