Curing HIV infections remains one of biomedicine’s most formidable challenges, in part because cells containing viral DNA in their chromosomes persist in the face of powerful drugs and immune responses. Now, for the first time, a research team has isolated individual 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 highlighted the result as one of the most innovative presented at the 24th International AIDS Conference that began the 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 reconstituted his immune system with cells impervious to HIV infection.
Efforts to develop simpler, safer cures for the other 38.4 million people living with the virus have been dogged by a fundamental obstacle: HIV persists in silent pockets of cells. After entering a human cell and integrating its DNA into the host’s 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 with the CD4 receptor harbor latent HIV DNA.
Researchers have used various compounds in what’s called a shock-and-kill strategy, which wakes up hidden viruses and directly destroys host cells or lets the immune system do the dirty work. This, in theory, should powerfully reduce or even eliminate the remaining reservoirs. But people who stop antiretrovirals after receiving these compounds routinely have HIV skyrocket to high blood levels within weeks.
At the AIDS conference, Eli Boritz, an immunologist at the National Institute of Allergy and Infectious Diseases (NIAID), described his team’s effort to better understand HIV’s hiding places by analyzing cells individual cells with the viral DNA in a latent state. Previous studies have isolated HIV within individual reservoir cells, but scientists couldn’t assess the host cell’s gene activity because of a Catch-22: They could only identify whether a cell was infected by prompting the virus to copy itself, which in turn likely altered cellular gene expression.
The new work sidestepped that dilemma by using a technique that isolates individual infected cells as small amounts of blood move through three microfluidic devices developed by physicist Adam Abate of the University of California, San Francisco, and bioengineer Iain Clark from UC Berkeley. In essence, the devices push blood through channels in microchips that trap individual cells in droplets, allowing them to be sliced 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 individual latently infected CD4 cells from three HIV-positive people with CD4 cells from three uninfected people. When a gene is activated, 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 cells in the reservoir inhibited signaling pathways that normally lead to cell death, and they also activated genes that silenced the virus itself.
“It’s remarkable that these cells are so different,” 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 he is already exploring the genes Boritz’s team identified and wonders whether a genome-editing method like CRISPR could destroy the reservoirs by, say, crippling one of the CD4 genes that are blocking their cell death pathway.
Lichterfeld says his lab has unpublished work that similarly suggests that these infected reservoir cells have special properties that make them resistant to immune attack. “It’s actually really nice how we used totally different technological approaches but came to relatively similar conclusions,” he says.
Boritz, whose group spent 11 years on this project, says the results make “perfect sense for this nebulous phenomenon we theorize about called virus latency.” It is 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 infection longer than others. Or it could be that HIV infection turns cells into long-lasting bunkers. “It’s extremely important for us to flesh that out,” says Boritz. “Maybe we could inhibit this mechanism.”