Curing HIV infections remains one of the most formidable challenges in biomedicine, in part because cells that retain the viral DNA in their chromosomes persist despite potent drugs and immune responses. A research team has now for the first time isolated single cells from these persistent viral reservoirs and characterized their gene activity, suggesting possible new healing strategies.
“This is really exciting,” said Sharon Lewin, head of the Peter Doherty Institute for Infection and Immunity, calling the result one of the most groundbreaking results presented at the 24th International AIDS Conference that began last week. “This single-cell advancement is great.”
AIDS researchers have had many victories since the disease emerged 42 years ago, but only four people are considered cured and they had cancers that required high-risk bone marrow transplants. The transplants re-established their immune systems with cells that were refractory to HIV infection.
Attempts to develop simpler and safer treatments for the other 38.4 million people living with the virus have been haunted by a fundamental obstacle: HIV stays in small cells by becoming silent. After 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 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 several compounds in a so-called shock-and-kill strategy, which wakes up the hidden viruses and either directly destroy the host cells or let the immune system do the dirty work. This should, in theory, forcefully reduce or even eliminate the remaining reservoirs. But people who stop taking antiretrovirals after routinely receiving these compounds have high 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 in individual cells in the reservoir, but scientists were unable to evaluate the host cell’s gene activity because of a Catch-22: They could only determine whether a cell was infected by prompting the virus to copy itself, which, in in turn, likely altered cellular gene expression.
The new work circumvented this dilemma by using a technique that isolates single, 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 of UC. Berkeley. Essentially, the devices push the blood through channels in microchips that trap individual cells in droplets, allowing them to be cut open for other instruments to read their genetic material.
“That’s a technology that didn’t exist before” for HIV research, says Mary Kearney, an HIV/AIDS researcher dedicated to 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 colleagues 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) that is used to make a protein. In their CD4 cell comparison, the researchers analyzed the entire array of nearly 18,000 mRNAs — the transcriptome — and found two distinct patterns: The CD4 cells in the reservoir inhibited signaling pathways that typically cause cell death, and they also activated genes that silence the virus itself. brought.
“It’s remarkable that these cells are so different,” said Mathias Lichterfeld, an infectious disease physician at Brigham and Women’s Hospital who studies HIV reservoirs in people who have had their infections under control for decades without treatment.
Lewin says she’s already sifting through the genes Boritz’s team identified and wondered if a genome-editing method like CRISPR could destroy reservoirs by, say, one of the CD4 genes that block the 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 attacks. “It’s actually really nice how we’ve used completely different technological approaches but come to relatively similar conclusions,” he says.
Boritz, whose group spent 11 years working on this project, says the results “make perfect sense for this fuzzy phenomenon we theorize about virus latency.” He is especially curious about what creates these patterns of gene expression. These CD4 cells may be different types with special properties that allow them to survive an infection longer than others. Or it could be that the HIV infection turns the cells into long-lasting bunkers. “It’s extremely important for us to figure that out,” Boritz says. “Maybe we can inhibit that mechanism.”