Single-cell studies offer a new perspective on how HIV infection persists — and potentially is cured |  Science

Single-cell studies offer a new perspective on how HIV infection persists — and potentially is cured | Science

Curing HIV infection remains one of the greatest challenges in biomedicine, in part because cells that contain the viral DNA in their chromosomes persist in the face of potent drugs and immune responses. A research team has now, for the first time, isolated individual cells from these stubborn virus reservoirs and characterized their gene activity, pointing to potential new healing strategies.

“This is really exciting,” says Sharon Lewin, who directs the Peter Doherty Institute for Infection and Immunity and singled out the finding as one of the most groundbreaking to be presented at the 24th International AIDS Conference that started last week. “These single-cell advances are big.”

AIDS researchers have celebrated many achievements since the disease emerged 42 years ago, but only four people are believed to have recovered, and they had cancer that required risky bone marrow transplants. The transplants reconstituted their immune systems with cells impermeable to HIV infection.

Efforts to develop simpler and safer cures for the other 38.4 million people living with the virus have been hampered by a fundamental obstacle: HIV persists in cellular pockets by becoming silent. After entering a human cell and integrating its DNA into the host chromosomes, HIV remains invisible to attack unless it starts making new viruses. Antiretroviral treatment suppresses HIV reproduction, but sensitive tests show that even with the most effective treatments, small populations of white blood cells occupied by the CD4 receptor harbor HIV DNA in a latent state.

Researchers have used various compounds in a so-called shock-and-kill strategy that wakes up the hidden viruses and either destroys the host cells directly or allows the immune system to do the dirty work. This should, in theory, greatly reduce or even eliminate any remaining reservoirs. But people who stop antiretroviral drugs after routinely receiving these compounds have spikes in blood levels of HIV 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 HIV’s hiding places by analyzing single cells with the viral DNA in a latent state. Previous studies have isolated HIV within individual cells in the reservoir, but scientists couldn’t assess the host cell’s gene activity because of a catch-22: They could only tell if a cell was infected by prompting the virus to copy itself what in turn probably changes cellular gene expression.

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

“It’s 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 many groups, including her own, will use it in the future.

Boritz and co-workers used the devices to compare the active genes in individual latently infected CD4 cells from three HIV-positive people with the CD4 cells from three uninfected people. When a gene is activated, its DNA is transcribed into a strand of messenger RNA (mRNA), which is used to make a protein. In their CD4 cell comparison, the researchers analyzed the entire suite of almost 18,000 mRNAs – the transcriptome – and found two distinct patterns: the reservoir CD4 cells inhibited signaling pathways that typically drive cell death, and they also activated genes that control the virus itself silenced.

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

Lewin says she’s already combing through the genes Boritz’s team identified and wonders if a genome-editing method like CRISPR could destroy reservoirs, for example by crippling one of the genes CD4 Genes that block its cell death pathway.

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

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

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