Doctors might soon be able to see the places where HIV replicates in the human body, if a technique used in macaques also works in people. A new method for imaging HIV in living macaques is described today in Nature Methods, a feat that had not been previously achieved in large, living animals. The finding is a big deal, because it means that researchers might one day be able to see — and selectively target — breeding grounds of HIV in people whose infection is undetectable in blood.

If you want to detect HIV in humans, blood is your friend. Unfortunately, the virus levels found in blood aren’t representative of the entire body because replication takes place in the cells located in tissues, not in blood. So, even though blood tests are perfectly adequate for detecting HIV in most people, they don’t always work. People on antiretrovirals sometimes have HIV levels that are so low that doctors can’t detect the virus in the bloodstream. In those cases, HIV continues to replicate in patients' organs, which limits doctors' ability to totally suppress the virus. Doctors could get around that issue by repeatedly sampling tissues at random and testing them for HIV replication — but doing that would seriously endanger their patients.

So, doctors are left with blood tests — and a bunch of unanswered questions. That’s why designing an imaging technique that can detect hidden pockets of HIV replication without doing anything too invasive is so important; it’s the missing methodological step that could help us figure out where HIV hides out in the body, and why it’s so damn persistent. "Total body imaging of sites of viral infections… will be revolutionary for the field of HIV therapeutics," — especially for therapies that focus on virus eradication and HIV remission, says Deborah Persaud, an HIV researcher at Johns Hopkins University who didn’t participate in the study.

In the study, researchers engineered a tracer labelled with radioactive copper that selectively attaches to a protein that’s only expressed on the surface of cells that produce HIV. Then, the researchers injected the tracer in macaques infected with SIV, the monkey form of HIV. They did the same thing with monkeys who had been on antiretrovirals for five weeks, and whose virus was undetectable. Approximately one day later, the researchers put the monkeys through a machine called a PET scan that can detect the low-level radioactivity coming from the tracer. The resulting images show HIV "hotspots" where the virus is making copies of itself — areas that researchers might one day be able to target therapeutically.

Results for an SIV-infected rhesus macaque (left) and an uninfected macaque (right). Credit: Santangelo et al., 2015

"We can find SIV activity in different organs," says Francois Villinger, a pathologist at Emory University and a co-author of the study. "And what’s especially interesting is that the signal is not uniform across organs; for example in the gastrointestinal tract, we found wide variability of signals." This means that small tissue samples that researchers sometimes take from animals infected with HIV might not be representative of the entire organ’s HIV replication levels, Villinger explains. "This is something that’s difficult to accept for some members in the field." Some scientists think that antiretroviral drugs should penetrate all parts of an organ equally, in which case the organ should be equally infected throughout. But "that doesn’t seem to be so," he says.

The researchers were also surprised to find important pockets of replication in the nose — in addition to signals in expected areas of replication like the gastrointestinal tract, the genital tract, and the lungs. "I don’t think it plays a role in transmission, but it’s interesting," Villinger says. "We found signals [in locations] that nobody has paid attention to."

The technique isn’t as sensitive as the researchers would like it to be. The tracer can still be detected organs that don’t experience replication because it’s in the process of being eliminated by these tissues. To get rid of these "background signals," the researchers injected non-HIV infected monkeys with the [tracer], and imaged them as well. Then, they subtracted their signals from those detected in HIV injected macaques. It worked out pretty well, Villinger says. But this correction isn’t ideal, as it might still lead to false positives, says Mathias Lichterfeld an HIV researcher at Harvard University who didn’t participate in the study. The researchers may be able to improve the sensitivity of the image by fiddling with the tracer — a crucial step in "getting the technique to a point where it can be used in humans," Lichterfeld says. Making the images more precise will also help the researchers image infected animals without using uninfected animals to exclude background effects.

The images also fall short when it comes to the brain. The tracer doesn’t penetrate the "blood-brain-barrier"— a selectively permeable barrier that separates the body’s blood from the central nervous system. As a result, the images don’t show viral replication in the brain, even though it’s probably taking place there too. This is unfortunate because a method that can detect replication in the central nervous system "is critically needed," Persaud says.

There’s one last issue with the method devised by Villinger’s team: it probably can’t detect latent HIV reservoirs, meaning pockets of HIV where replication doesn’t take place. This is a rare occurrence, but it does happen. In 2013, doctors announced that treating a baby with antiretrovirals right after birth had "functionally cured" the child of HIV; the virus remained undetectable after the child was taken off the drugs. Unfortunately, the virus returned a year later. And unfortunately, the protein to which the tracer binds isn't expressed in latent HIV reservoirs, Persaud says.

Now that the study has been published, Villinger and his team of researchers want to tweak the tracer to make the images more accurate. They also want to see if they can detect differences in HIV progression in animals infected intravenously — a model of infection through injection drug use — and animals infected through tissues in the genital tract. They would also like to measure signals in animals that have been on antiretrovirals therapy for longer periods, as well in animals that have been taken off antiretroviral therapy to see what happens as HIV replication is "rekindled." Villinger and his team have also started to look at what happens in the first two weeks of infection; scientists don't know much about that, he says. Finally, the scientists plan to try the method on humans — something that could happen in the next five years, Villinger says.

Being able to see reservoirs of HIV replication in living animals is a big deal. The system needs to be fine-tuned, and tested for safety, but if there’s progress, it would one day lead to a tool for monitoring the effectiveness of antiretroviral therapies, Villinger says. And in the meantime, it may give researchers a better idea of how HIV re-ignites in the body. "In my mind that might be the most important asset we can use this technology for," Villinger says. "It’s really trying to better define what happens where — and that’s really critical as we move forward in medicine."