Treating HIV infection remains one of the most difficult challenges in biomedicine, in part because cells that contain viral DNA in their chromosomes persist in the face of powerful drugs and an immune response. For the first time, the research team isolated individual cells from these stubborn viral reservoirs and characterized their gene activity, suggesting potential new treatment strategies.
“It’s really exciting,” said Sharon Levin, who heads the Peter Doherty Institute of Infection and Immunity, and called the results one of the most groundbreaking to be presented at the 24th International AIDS Conference, which opened last week. “These advances in single-cell production are big.”
AIDS researchers have had many triumphs since the disease was first discovered 42 years ago, but only four people are considered cured, and they had cancers that required risky bone marrow transplants. The transplants restored their immune systems with cells immune to HIV infection.
Efforts to develop simpler and safer drugs for the other 38.4 million people living with the virus are hampered by a fundamental obstacle: HIV remains silent in cells. After entering a human cell and integrating its DNA into the host’s chromosomes, HIV remains invisible to attack unless it starts producing new viruses. Antiretroviral treatment suppresses HIV reproduction, but sensitive tests show that even with the most effective treatments, small populations of white blood cells studded with the CD4 receptor contain latent HIV DNA.
The researchers used various compounds in a so-called “shock-and-kill” strategy, which awakens latent viruses and either directly destroys host cells or allows the immune system to do the dirty work. This should, in theory, greatly reduce or even eliminate any residual reservoirs. But people who stop antiretroviral therapy after regularly taking these compounds have a sharp rise in HIV levels 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 HIV’s hiding places by analyzing individual cells with latent viral DNA. Previous studies had isolated HIV inside individual cells in a reservoir, but the scientists couldn’t assess the gene activity of the host cell because of a Catch-22: they could only tell if the cell was infected, prompting the virus to copy itself, which in turn likely , changed the expression of cellular genes.
The new work avoided this dilemma by using a technique that isolates individual infected cells when tiny volumes of blood are passed through three microfluidic devices developed by physicist Adam Abate of UC San Francisco and bioengineer Ian Clark of UC Berkeley. Essentially, the devices push blood through channels in microchips that trap individual cells in droplets, allowing them to be cut open so other tools can read their genetic material.
“It’s a technology that didn’t exist before” for HIV research, says Mary Kearney, an HIV/AIDS researcher who focuses on reservoirs. Lillian Kohn, 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 his colleagues used the devices to compare active genes in individual latently infected CD4 cells from three HIV-positive people with CD4 cells from three uninfected people. When a gene is turned on, its DNA is transcribed into a strand of messenger RNA (mRNA), which is used to make a protein. When comparing CD4 cells, the researchers analyzed the entire set of nearly 18,000 mRNAs—transcripts—and found two clear patterns: CD4 reservoir cells suppressed signaling pathways that normally cause cell death, and they also activated genes that silenced the virus itself.
“It’s surprising 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 have managed their infections without treatment for decades.
Levine says she is already investigating the genes Boritz’s team identified and is wondering whether a genome-editing technique like CRISPR could destroy water bodies, such as by damaging one of the CD4 genes that block the pathway of cell death.
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 actually pretty 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 about called viral latency.” He is particularly interested in what creates these patterns of gene expression. Perhaps these CD4 cells are different types with special properties that allow them to survive the infection longer than others. Or HIV infection turns cells into long-lasting bunkers. “It’s extremely important for us to figure this out,” Boritz says. “Maybe we could put the brakes on that mechanism.”