A team of researchers studying HIV have created a test that they say will allow clinicians and scientists to better track efforts to cure the disease.
Most HIV drugs attack the active virus, which enters immune cells, replicates, and then buds off of the cell it has just crippled. But, after infection, HIV, which has an RNA genome, can also make a DNA copy of its RNA and insert that DNA into its host’s DNA, lurking as something called a provirus. A subsequent immunological trigger can prompt these proviruses to become active, forming viruses that can then attack immune cells.
These lurking proviruses are a key barrier to curing the disease. But current tests that measure the number of proviruses in someone who is HIV-positive can’t distinguish between highly mutated proviruses that would be harmless and ones that are more pristine, and therefore potentially more harmful should they be woken up, says Robert Siliciano of the Johns Hopkins University School of Medicine. He and his team now report a test that can make this important distinction (Nature 2019, DOI: 10.1038/s41586-019-0898-8).
Researchers are developing drugs that work against proviruses in an infected person, Siliciano says. “But before this work, there really wasn’t a good way to know whether these interventions were actually doing anything.”
In previous work, Siliciano’s team found that up to 98% of proviral DNA detected by a standard polymerase chain reaction assay was too mutated to ever form an infectious HIV particle. This standard test uses PCR to amplify all proviral DNA in a person’s genome for identification, no matter how mutated it is. In the new test, called the droplet digital PCR test, mostly proviral DNA with minimal mutations gets amplified, allowing scientists to focus more on potentially competent proviruses.
To do this, researchers parse sample DNA from a person into nanodroplets so that each droplet contains only one provirus. They then run PCR reactions in each nanodroplet in such a way that the amplification fails or is less robust when there are significant numbers of mutations present.
In tests using small quantities of cells from HIV-infected people who are receiving HIV treatment, the research team could detect and distinguish intact proviral DNA from its mutated counterparts.
“It’s much better than the previous assay,” says David Margolis, an HIV expert at the University of North Carolina at Chapel Hill, adding that it is still not exact. Up to 30% of the proviruses detected with the method still have mutations. Despite that, Margolis thinks the test could aid efforts to study drug candidates targeting proviruses.
One of Siliciano’s former students has spun a company to further develop this technology.