Curing HIV infections remains one of the most daunting challenges in biomedicine, in part because cells carrying the viral DNA in their chromosomes persist in the face of powerful drugs and immune responses. A research team has now, for the first time, isolated single cells from these stubborn viral reservoirs and characterized their gene activity, suggesting potential new treatment strategies.

“This is really exciting,” says Sharon Lewin, who directs the Peter Doherty Institute for Infection and Immunity and singled out the result as one of the most innovative presented at the 24th International AIDS Conference which began last week. “These single-cell advances are huge.”

AIDS researchers have had many triumphs since the disease emerged 42 years ago, but only four people are considered cured, and they had cancers that required risky bone marrow transplants. The transplants rebuilt their immune systems with cells impervious to HIV infection.

Efforts to develop simpler and safer cures for the other 38.4 million people living with the virus have been stymied by a fundamental obstacle: HIV persists in silent pockets of cells. Once it enters a human cell and integrates its DNA into the host’s chromosomes, HIV remains invisible to attack unless it starts producing new viruses. Antiretroviral treatment suppresses HIV replication, but sensitive tests show that even with the most effective treatments, small populations of white blood cells loaded with the CD4 receptor carry HIV DNA in a latent state.

Researchers have used various compounds in what’s called a hit-and-kill strategy, which wakes up hidden viruses and either destroys host cells directly or allows the immune system to do the dirty work. This should, in theory, greatly reduce or even eliminate any remaining reservoir. But people who stop antiretrovirals after taking these compounds routinely have high levels of HIV in their blood within weeks.

At the AIDS conference, Eli Boritz, an immunologist at the National Institute of Allergy and Infectious Diseases (NIAID), described his team’s efforts to better understand the reservoirs of HIV by analyzing single cells with viral DNA. in a latent state. Previous studies have isolated HIV inside single cells in the reservoir, but scientists couldn’t assess the host cell’s gene activity because of a Catch-22: They could identify whether a cell had been infected by prompting the virus to copy itself, which, in turn, is likely to alter cellular gene expression.

The new work avoided this dilemma by using a technique that isolates single, infected cells while moving small amounts of blood through three microfluidic devices developed by physicist Adam Abate at the University of California, San Francisco and bioengineer Iain Clark at UC Berkeley. Essentially, the devices push blood through channels in microchips that capture individual cells in droplets, allowing them to open up so that other instruments can read their genetic material.

“This is a technology that didn’t exist before” for HIV studies, says Mary Kearney, an HIV/AIDS researcher who focuses on reservoirs. Lillian Cohn, who studies HIV reservoirs at the Fred Hutchinson Cancer Research Center, says developing this new technology required a “heroic effort” and predicts that many groups, including her own, will use it in the future.

Boritz and colleagues used the devices to compare the active genes in latently infected individual CD4 cells from three HIV-positive people with the CD4 cells of three uninfected people. When a gene is turned on, its DNA is transcribed into a strand of messenger RNA (mRNA) that is used to make a protein. In their comparison of CD4 cells, the researchers analyzed the entire pool of nearly 18,000 mRNAs—the transcriptome—and found two distinct patterns: CD4 reservoir cells inhibited signaling pathways that normally trigger cell death, and they also activated genes that silence the virus itself.

“It’s remarkable that these cells are so distinct,” says Mathias Lichterfeld, an infectious disease physician at Brigham and Women’s Hospital who studies HIV reservoirs in people who control their infections for decades without treatment.

Lewin says she is already cleaning up the genes that Boritz’s team identified and is wondering whether a genome-editing method like CRISPR could destroy the reservoirs by, for example, crippling one of the CD4 genes that are blocking its cell death pathway.

Lichterfeld says his lab has unpublished work that similarly suggests these infected reservoir cells have special properties that make them resistant to immune attack. “It’s really cool how we used completely different technological approaches but came to relatively similar conclusions,” he says.

Boritz, whose group spent 11 years on the project, says the results make “perfect sense for this nebulous phenomenon we theorize called virus latency.” He is particularly curious about what creates these patterns of gene expression. It may be that these CD4 cells are distinct types with special properties that allow them to survive infection longer than others. Or it could be that HIV infection transforms cells into long-term bunkers. “It’s extremely important for us to do this,” says Boritz. “Perhaps we could inhibit that mechanism.”

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