Curing HIV infections remains one of the most formidable challenges in biomedicine, in part because cells that hold 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 by their gene activity, suggesting potential new cure strategies.
“This is really exciting,” says Sharon Lewin, who heads the Peter Doherty Institute for Infection and Immunity and singled out the result as one of the most groundbreaking presented at the 24th International AIDS Conference that began last week. “These single-cell advances are big.”
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 risk-laden bone marrow transplants. The transplants reconstituted 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 dogged by a fundamental obstacle: HIV persists in pockets of cells by going silent. After entering a human cell and integrating its DNA into the host chromosomes, HIV remains invisible to attack unless it starts to produce new viruses. Antiretroviral treatment squelches HIV reproduction but sensitive tests show that even with the most effective treatments, small populations of white blood cells studded with the CD4 receptor harbor HIV’s DNA in a latent state.
Researchers have used various compounds in what’s called a shock-and-kill strategy, which wakes up the hidden viruses and either destroys the host cells directly or allows the immune system to do the dirty work. This, in theory, should powerfully reduce or even eliminate any 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 hideouts by analyzing single cells with the viral DNA in a latent state. Previous studies have isolated HIV inside of single cells in the reservoir, but scientists could not evaluate the host cell’s gene activity because of a Catch-22: They could only identify whether a cell was infected by prodding the virus to copy itself, which, in turn, likely altered the cellular gene expression.
The new work added this dilemma by using a technique that isolates single, infected cells as tiny amounts of blood move through three microfluidic devices developed by physicist Adam Abate at the University of California, San Francisco, and bioengineer Iain Clark at UC Berkeley. In essence, the devices push the blood through channels in microchips that trap individual cells in droplets, allowing them to be cut open so that other instruments can read their genetic material.
“That is a technology that did not previously exist” 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 this 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 of three uninfected people. When a gene turns on, its DNA is transcribed into a strand of messenger RNA (mRNA) that is used to make a protein. In their CD4 cell comparison, the researchers analyzed the entire suite of nearly 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 silenced the virus itself.
“It’s remarkable that these cells are so distinct,” says Mathias Lichterfeld, an infectious disease clinician at Brigham and Women’s Hospital who studies HIV reservoirs in people who control their infections for decades without treatment.
Lewin says she’s already scouring the genes that Boritz’s team identified and wondering whether a genome-editing method such as CRISPR could destroy reservoirs by, for example, crippling one of the CD4 genes that is 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 actually really nice how we used totally different technology approaches but reached 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.” He’s particularly curious about what creates these patterns of gene expression. It could 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 the HIV infection transforms the cells into long-lasting bunkers. “It’s extremely important for us to piece that out,” Boritz says. “Perhaps we could inhibit that mechanism.”