Advances in HIV Research May Uncover Viral Reservoirs

Research published in Molecular Therapy in early May could help uncover latent HIV-1 cells that serve as a viral reservoir.

Researchers from the Lewis Katz School of Medicine (LKSOM) at Temple University and the University of Pittsburgh, Pennsylvania, used gene editing technology to excise HIV DNA from the genomes of 3 animal models. Their findings were published in Molecular Therapy on May 3, 2017.

For years, the HIV virus was regarded as a death sentence. That is, until antiretroviral therapy (ART) was made available to those with infection in 1996. Now, however, HIV-associated death rates are at an all-time low. According to a recent study that examined survival rates among HIV-positive individuals living in Europe and the United States, the decline in HIV-related death rates has been consistent across the continents, and in subjects of different age groups, and CD4 cell count. In 1996 death rate had been approximately 6%, whereas in 2010 the death rate went down to 3%. The researchers attribute this to the development of more effective ART regimens.

It seems that HIV may have been stealthier than originally believed. Recently, researchers from the University of North Carolina School of Medicine identified an additional reservoir in the form of macrophages.

Now, researchers from LKSOM are working to bring HIV to its knees and uncover these latent cells. The team was able to completely shut down HIV-1 replications infected into the animals. They successfully eliminated the infected cells with clustered regularly interspaced short palindromic repeat (CRISPR) technology, and CRISPR associated protein 9 (Cas9).

Included in their successful study was a “humanized” model — a mouse transplanted with human immune cells and infected with the virus. It is the first evidence of HIV-1 eradication of prophylactic treatment with a CRISPR/Cas9 system.

According to a press release, the study was a follow-up on the team’s published proof-of-concept study from last year, when they incorporated HIV-1 DNA into the genome of every tissue of transgenic rat and mouse models, and demonstrated how they could delete the fragments of HIV-1 from the genome in most of the subjects’ tissues.

Lead researcher, Wenhui Hu MD, PhD, associate professor in the Center for Metabolic Disease Research and the Department of Pathology at LKSOM, said the most recent study is more comprehensive, and confirms their previous data while also improving their gene editing efficiency.

“We also show that the strategy is effective in 2 additional mouse models, one representing acute infection in mouse cells and the other representing chronic, or latent, infection in human cells,” Hu said. The team was able to genetically inactivate HIV-1 in transgenic mice, and reduce the RNA expression of viral genes by 60 to 95%.

When testing their methods with mice acutely infected with EcoHIV — the mouse equivalent of HIV-1 – results were similarly successful despite the HIV actively replicating. The team tested CRISPR’s ability to block viral replication and possibly prevent systemic infection, achieving 96% excise efficiency in mice with EcoHIV.

The final form of animal model infection included engrafting mice with human immune cells and T-cells, then infecting them with latent HIV-1. Though the latent virus is capable of escaping detection in human T-cells, a single CRISPR/Cas9 treatment excised viral fragments from human cells embed in the mouse tissues and organs.

The team was able to measure HIV-1 RNA levels with a bioluminescence imaging system, developed by Won-Bin Young, PhD, assistant professor in the Department of Radiology at the University of Pittsburgh School of Medicine at the time.

The imaging system made it possible for them to view the HIV-1-infected cells in the body and observe their replication in real-time. The team was also able to view HIV-1 reservoirs in latent cells and tissues — the infection’s strongest asset to eluding treatment.

Kamel Khalili, PhD, Team member and director of the Center for Neurovirology and director of the Comprehensive NeuroAIDS Center at LKSOM, said the next stage would be to apply the study to nonhuman primates, “a more suitable animal model where HIV infection induces disease.”

Researching the CRISPR/Cas9 treatment’s effect on primates would include testing more latent T-cells and sanctuary sites for HIV-1 — including brain cells. The study is working towards human application.

“Our eventual goal is a clinical trial in human patients,” Khalili said.