A new tool called HIV-seq is giving scientists their clearest view yet of HIV-infected cells that linger despite treatment. The findings could guide future strategies to calm or clear these long-lived reservoir cells.
For decades, doctors have relied on antiretroviral therapy to keep HIV under control, turning what was once a fatal infection into a manageable chronic condition. But even when the virus is suppressed to undetectable levels in the blood, a small population of infected immune cells persists in the body, quietly harboring HIV.
Those cells, known as the HIV reservoir, are the main reason treatment must continue for life. If therapy stops, the virus can roar back.
Now, scientists at Gladstone Institutes and the San Francisco Veterans Affairs Medical Center have developed a powerful new tool, called HIV-seq, that reveals what those reservoir cells are actually doing inside people on and off treatment — and why they are so hard to eliminate.
The work, published in Nature Communications, suggests that many of these cells are not as silent as once thought and may actively produce viral fragments that fuel long-term inflammation and health problems.
Senior author Nadia Roan, a senior investigator at Gladstone, noted the field has long described the reservoir as “latent,” implying that the virus inside these cells is completely inactive.
“But notion that the entirety of the HIV reservoir is latent is actually a misleading description, because some reservoir cells can still be quite active,” Roan, also a professor in the Department of Urology at the University of California San Francisco, said in a news release. “Even though antiretroviral therapy keeps full-fledged HIV virus from being made, some of the infected cells continue spitting out viral products.”
Those viral remnants do not cause full-blown infection while therapy is working, but they can keep the immune system in a constant state of low-level alarm. Over years, that chronic inflammation is linked with organ damage, higher risk of heart attack and other complications in people living with HIV.
Researchers have long suspected that understanding which genes are switched on inside these reservoir cells could point to new ways to either shut them down or remove them altogether. The challenge has been finding and analyzing enough of these rare cells in blood samples from people whose HIV is suppressed.
In recent years, single-cell RNA sequencing — a technique that reads which genes are active in individual cells — has transformed many areas of biology. But it has struggled with HIV reservoir cells in treated patients.
“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,” added co-first author Julie Frouard, a scientist in the Roan lab. “That’s not enough for a meaningful analysis.”
The problem comes down to the virus’s genetic material. Standard single-cell RNA methods are tuned to capture certain types of RNA fragments. Much of the RNA produced by HIV does not fit those criteria, so infected cells that are actively making viral RNA can be missed.
To solve that, the team designed HIV-seq specifically for HIV. The new method still profiles gene activity one cell at a time, but it is engineered to better recognize and capture HIV RNA fragments alongside human RNA. That makes it far more likely to spot infected cells, especially those that are actively producing viral material.
“Pitting HIV-seq head-to-head with the standard approach, we recovered and analyzed more HIV-infected cells, and higher numbers of HIV RNA within those infected cells,” added co-senior author Steven Yukl, a physician-scientist at the San Francisco VA Medical Center. “Now, for the first time, we can actually characterize these cells in a meaningful manner for people whose HIV is suppressed by antiretroviral therapy.”
Using HIV-seq, the researchers were able to recover 25 reservoir cells from three people on antiretroviral therapy — a substantial improvement over earlier efforts. In people with active HIV infection who had not yet started therapy, the tool identified more than 1,000 infected cells from four patients, the highest number reported so far with this kind of single-cell analysis.
With those larger cell sets in hand, the team compared HIV-infected cells from people before and after they began treatment and looked at the proteins on the cells’ surfaces.
“Prior single-cell RNA sequencing studies have primarily analyzed HIV-infected cells in people who had not yet started therapy,” added co-first author Sushama Telwatte, a former postdoctoral researcher in the Yukl lab and now an investigator at the Doherty Institute, University of Melbourne. “We felt those cells probably look very different from reservoir cells in people on therapy, which can persist for decades while still producing HIV RNA fragments.”
The comparison confirmed that suspicion. In people who had not started therapy, infected cells showed cytotoxic features — they carried proteins linked with the ability to kill other cells. They also had lower levels of certain genes associated with suppressing HIV, suggesting the virus may dial down those defenses to churn out more copies of itself.
“In a general sense, I would say that these cells were rather inflammatory, or fiery,” Roan added.
In contrast, reservoir cells from people on long-term therapy looked much calmer. They lacked those cytotoxic traits and instead showed anti-inflammatory features. They also had higher levels of genes that help cells resist death and survive for long periods.
That survival advantage may be part of what allows reservoir cells to linger for decades, even when the virus they carry is held in check by drugs. The team also found higher levels of other proteins in treated patients’ reservoir cells, including one linked with the ability to keep dividing over time and others tied to suppressing both HIV production and the immune response.
Those patterns could help explain how these cells manage to fly under the immune system’s radar while continuing to produce viral fragments.
The findings also intersect with ongoing clinical efforts.
“This is noteworthy because there is an ongoing clinical trial testing a drug targeting a pathway that HIV may use to preferentially promote survival of its host cell,” added Yukl, who is also a professor of medicine at UCSF. “Our data provide further support for that research.”
By highlighting the gene pathways and surface markers that distinguish “fiery” pre-therapy cells from quieter, long-lived reservoir cells on treatment, HIV-seq offers a roadmap for future interventions. Drugs might be designed to block the survival pathways that reservoir cells rely on, or to make them more visible to the immune system.
“Using our new tool, we’ve found key differences in people’s HIV-infected cells before versus after starting antiretroviral therapy,” Roan added. “We hope it will be helpful for understanding how HIV develops, and how the long-lived HIV reservoir can persist for decades in people with HIV.”
Her team is already moving from observation to experimentation.
“We’re already building on some of our new findings by testing, in various laboratory models, whether we can stop HIV reservoir cells from multiplying by targeting these pro-survival pathways,” added Roan. “We hope this is just the beginning of all that could be discovered with HIV-seq.”
While a complete cure for HIV remains elusive, tools like HIV-seq are giving scientists a much sharper picture of the virus’s last strongholds. By exposing how reservoir cells change with therapy and what keeps them alive, this work brings the field a step closer to strategies that might one day free people from lifelong treatment.
Source: Gladstone Institutes