Youry Kim
Infectious Disease | dPCR

Unlocking a real cure for HIV: Viral shock and kill therapy

Ever since the Human Immunodeficiency Virus (HIV) was first identified in the early 1980s, researchers have been searching in vain for a cure. While antiretroviral therapy (ART) has helped millions live healthy lives with the infection, HIV and AIDS burden health care systems and people continue to suffer from social stigma. Is a cure on the horizon?
Back in 2011, curing people of Human Immunodeficiency Virus seemed a long shot. HIV is a retrovirus - it integrates its own DNA into the DNA of human cells and sometimes goes dormant, hiding out from the immune system and the medicines researchers have developed to kill it. 

Youry Kim was still an undergraduate studying biomedical science at Monash University at the time, when a series of lectures by Sharon Lewin, director of the Peter Doherty Institute for Infection and Immunity in Melbourne, Australia, piqued her interest in the sly tactics that HIV uses to thwart the human immune system. 

When Lewin offered Kim the opportunity to do an honors project in her lab studying latent HIV, Kim jumped at the chance. The project led to a Ph.D. under Lewin’s tutelage. 

Today Kim is a post-doc in Lewin’s lab, focused on finding drugs that can eliminate latent HIV from human cells so people living with HIV are not reliant on antiretroviral therapy (ART) for the rest of their lives. 

ART revolutionized HIV and AIDS treatment when it first became available in the mid-1990s, but HIV is still a major issue globally. The therapy's powerful ability to suppress viral replication, and thereby enable the immune system to recover, gave hope that at the very least, the infection could be managed long-term. But ART is not a cure.

In middle-and low-income countries, up to 30% of people living with HIV have limited access to ART, says Lewin, so the number of the newly infected continues to grow. And despite the success of Antiretroviral Therapy in both treating and preventing HIV, the infection remains a public health burden.  
In middle-and low-income countries, up to 30% of people living with HIV have limited access to antiretroviral therapy (ART), says Lewin. Interruption of ART can reactivate the virus, causing rebound and immune damage. This means the number of newly infected continues to grow. U.N. AIDS estimates that low-and middle-income countries spent US$ 21 billion on global AIDS response in 2021 but that $29 billion will be required in 2025 to get on track to end AIDS as a global public health threat. (1) The psychological burdens and social stigma associated with the necessity of lifelong treatment and the potential for viral rebound serve to exacerbate the complexities linked with latent HIV.
Only around one in one thousand CD4 cells is infected with latent HIV in people taking ART and only a tiny fraction of that pool contains intact HIV DNA.
Youry Kim, Peter Doherty Institute of Infection and Immunity, University of Melbourne in Australia

How HIV hides from the immune system

Once the virus infects human immune cells, it uses a number of strategies to evade the immune system. For one, HIV replicates and mutates at lightning speed, making it tough for the immune system to adapt quickly enough to fight it. In addition to infecting active immune cells, the virus also infects resting immune cells, where it goes dormant and hides.

These HIV reservoirs mainly consist of a type of immune cell called resting CD4+ T cells, which behave like sleeper cells - they don’t actively produce new virus nor do they trigger an immune response.

This makes it harder for the immune system and medications to detect and eliminate them and is also the reason why in as little as 2 to 3 weeks after a person stops ART, HIV levels rebound: Dormant virus becomes active again and the cycle of replication, reinfection and destruction of new CD4 cells continues.

Advances in our understanding of HIV’s behavior and the availability of new technology and medicines from the cancer field have rekindled hopes that researchers will be able to develop a functional cure for people living with HIV - one that eliminates or slashes levels of latent HIV enough so that the immune systems of people living with HIV will be able to control the virus without life-long antiretroviral medication.

A cure would not only save health care systems billions spent on life-long ART for the roughly 40 million people currently living with HIV, but it would also eliminate the social stigma that many of them suffer, says Lewin.

It would also offer a safer alternative since studies have shown that people taking ART long-term have a higher risk of many ailments that include cardiovascular, liver and kidney disease.(2)

“This is what is driving people around the world trying to find a cure,” Lewin says. 

Latent HIV hides in immune cells and tissues - HIV reservoirs - evading both the immune system and standard drugs. HIV reservoirs can remain dormant and persist despite treatment, primarily in the form of latent reservoirs like resting CD4+ T cells and tissue macrophages. Individuals with latent HIV can also unknowingly transmit the virus, emphasizing prevention and regular testing.
You really want to be targeting replication competent intact provirus.
Youry Kim, Peter Doherty Institute of Infection and Immunity, University of Melbourne in Australia

Using cancer treatments for HIV

For years, the viral craftiness of HIV had dampened hopes that a cure was even possible. But then, in 2007, came news that a bone marrow transplant cured a person with HIV. The patient, Timothy Ray Brown, was HIV positive and suffering from leukemia. The donor marrow came from a person who was naturally resistant to HIV infection.  Brown became the first person known to be cured of HIV - there were no detectable levels of virus in his blood.

While a bone marrow transplant for people living with HIV was never considered a desirable or even practical cure because of the health risks from the procedure and the expense, Brown’s case showed that a cure was possible.

Then came another unexpected boon to the field: Targeted cancer treatment. About a decade ago, a class of new cancer drugs became available that blocked cancer’s ability to “hide” from the immune system. The advance got HIV researchers thinking about whether they might use the same approach to target latent HIV. Although cancer is a distinct disease, researchers discovered that both cancer and HIV share certain behaviors in common, “which is why a lot of these cancer drugs have been repurposed for HIV,” says Kim.

Mutations in cancerous cells, for example, are thought to alter the structure of chromatin - material that forms chromosomes - in cancer cells, making it easier for them to proliferate.  The ability of HIV to integrate into host cell DNA, researchers think, is also influenced by how chromatin is organized. In the cancer field, there are many drugs that work by loosening chromatin to make it more accessible to cellular machinery to initiate transcription, says Kim, thereby tamping down the cell’s ability to replicate. Researchers theorized that these same cancer drugs might also force cells infected with latent HIV to initiate transcription of viral DNA, making them “visible” to the immune system.

However, while the approach could trigger latent HIV to replicate, it didn’t prove powerful enough to rid the body of infected cells. Researchers then took a cue from another similarity between cancer and HIV. Cells infected with HIV have high levels of specific proteins that may tip the cells towards survival, outliving their normal lifecycle that would include programmed cell death, or apoptosis - some cancer cells produce the same proteins and circumvent apoptosis too. Drugs that inhibit these proteins are already available to treat cancer.

In the early 1990s, researchers discovered a gene mutation that appeared to make people resistant to HIV infection: CCR5-delta32. This rare delta32 mutation makes it difficult for HIV to latch onto cell receptors, hindering the virus’ ability to infect cells (However, people with CCR5-delta32 can still become infected with HIV strains that use alternate co-receptors). Timothy Ray Brown, also known as the Berlin patient, received a bone marrow transplant from a donor with the CCR5-delta32 mutation. Following the transplant, Brown stopped ART and remained in remission until his death in 2020 from cancer.
The goal is to “shock” latent HIV out of hiding and then “kill” the infected cells.
Youry Kim, Peter Doherty Institute of Infection and Immunity, University of Melbourne in Australia

The “shock and kill” approach to curing HIV

In what’s called the “shock and kill” approach to curing HIV, Kim is now focusing her efforts in combining a drug to trigger latent HIV to replicate, a so-called “latency-reversing” agent, with a pro-apoptotic drug to first “shock” latent HIV out of hiding and then “kill” the infected cells.  

To test the approach, Kim treats blood samples from people living with HIV with the drug combination and compares them to untreated blood samples from the same person. Using the QIAcuity Digital PCR System, she then measures levels of integrated HIV DNA in both treated and untreated samples.

The challenge had been determining whether the drugs are eliminating intact virus. Not all latent HIV is “replication-competent,” Kim explains.

Because the virus mutates so quickly, 90% of HIV DNA is defective and therefore can’t replicate, it is unable to infect CD4 cells. With the drug combination, “you really want to be targeting replication competent intact provirus.” This is made even more difficult by its scarcity. “It’s only a tiny fraction,” she adds. “Only around one in one thousand CD4 cells is infected with latent HIV in people taking ART and only a tiny fraction of that pool contains intact HIV DNA,” she says.

Kim now has optimized her workflow to get more meaningful results and to save time as well.  For DNA extraction, for example, she uses QIAGEN QIAamp DNA extraction kits, and QIAGEN AllPrep kits to extract both DNA and RNA. Previously she was using the Allprep kits for DNA extraction too. But she found that the assay tended to inflate the estimated amount of DNA present in her sample. The QIAamp kit for DNA extraction proved more reliable, she says.

Nucleic acids in the samples are then amplified using QIAcuity. “We decided to use QIAcuity for several reasons,” she says. For one, hands-on time is vastly reduced, she explains. “This is because the QIAcuity divides the sample into thousands of partitions in a nanoplate, and does the PCR and imaging steps all in one machine. Other systems require two to three different machines and the time to perform the same experiment is hours longer,” she adds. With QIAcuity, Kim can also include more than two probes in one mixture (dPCR multiplexing), which reduces the amount of nucleic acid required for her experiments.

Youry Kim is a post-doctoral Research Fellow at the Peter Doherty Institute of Infection and Immunity at the University of Melbourne in Australia. Kim’s research focuses on finding new drug compounds to reactivate latent HIV and eliminate infected cells. Kim has always been interested in science, she says. After hearing lectures in immunology about how cells respond to infection, she was hooked on studying infectious disease. “I want to understand how the body responds to infection, down to the cellular level.”
Measuring the amount of intact virus helps us see how well our treatments are working.
Youry Kim, Peter Doherty Institute of Infection and Immunity, University of Melbourne in Australia

Still looking for the right combination

Using a multiplex dPCR assay that Kim and her colleagues adapted to the QIAcuity system, she can now detect and quantify the fraction of intact HIV DNA in her samples.  “Measuring the amount of intact virus helps us see how well our treatments are working,” she adds.

Researchers working on the shock and kill approach continue to look for drug combinations that might have the desired effect.

To reduce toxicity, some researchers are also looking at delivering shock and kill drugs with more precision - packaging them into nanoparticles that hone specifically to infected cells, for example.

Scientists are also pursuing other ways to cure HIV. Among them are the “block and lock” approach that aims to permanently silence latent virus with drugs that block the transcription step in the cell's lifecycle. Over time, this would cripple HIV’s ability to replicate. Still others are studying gene therapy approaches that could knock out genes that enable the virus to infect cells.

In the meantime, Kim is hoping that the positive results she is seeing in the lab will lead to clinical trials to test the efficacy of her drug combination in people living with HIV. But of course, she adds: “Any approach that leads to a cure for HIV is wanted.”

Elite controllers are also a major part of finding a cure. These are a fraction of individuals with HIV who have managed to maintain undetectable viral loads naturally. One woman, 66-year-old Loreen Willenberg, has been living with HIV since 1992, is not taking ART, and yet shows no detectable levels of HIV in her blood. Scientists are still trying to understand how this group of elite controllers are able to keep the virus in check.