Roundabout to Remission

Steve Deeks is peripatetic by nature: probably a good quality, given the challenges in HIV cure research. The biggest is HIV persistence, the subject of a recent symposium.

By Michael Dumiak

We would like treatment that we can stop.

This is the answer Institut Pasteur scientist and Nobel Laureate Francoise Barré-Sinoussi hears when she asks people living with HIV what they expect from scientists today. Barré-Sinoussi raised a smoldering question in HIV cure research as keynote speaker at the Keystone Symposium,Mechanisms of Persistence: Implications for a Cure, held in Boston April 24-May 1. What will it take for HIV-infected individuals to be able to interrupt antiretroviral (ARV) therapy and achieve a sustained remission from the virus? 

This long-lived remission may be the field’s best hope. But getting to that point may require understanding a vast amount of unsurveyed territory, starting with where to find and how to measure the reservoir of latent HIV-infected cells that forms soon after infection and persists even during suppressive ARV treatment.

Scientists are experimenting with ways to activate and ultimately flush out this viral reservoir. They are also hunting for markers they can use to measure the success of these efforts and are deciding when to study cure strategies in clinical trials that will require volunteers to temporarily stop treatment.

Interrupting ARV at this point in cure research is controversial. “We are not curing people. So why stop therapy?” asks Steve Deeks, a clinician at the University of California in San Francisco (UCSF) and an expert in the role of chronic inflammation. He concedes that this is a reasonable objection, but says the counter-argument is that a cure will never happen unless researchers test multiple strategies. “Many of us believe the best way to do this is in the context of treatment interruption studies,” he says. “The interruptions can be done safely, if people are monitored carefully.”

Deeks is an early riser, awake by 4:30 or 5AM. By mid-morning he is ready to hit the gym and work out some of the boundless energy he’s shown over the last week as co-host of the Keystone Symposium. He has a unique perspective on and long history in HIV research. He is a manager of one of the largest and long-lived cohorts of HIV-infected men and women in the world—a highly valuable living and breathing data set. Barré-Sinoussi tapped Deeks five years ago to help create a strategic framework for HIV cure research.

At Keystone he eagerly jumped into discussions on the pros and cons of studies, encouraging some researchers to go further, challenging others on their findings. But he’s reticent to discuss himself: Deeks says it’s not in his phenotype to sit and talk, and he quickly gets bored and restless. “I don’t do exciting things. I ride my bike,” Deeks says. But in San Francisco, which is a very hilly place. “I have an electric bike,” he counters. It is with this droll humor that Deeks steers IAVI Report through the current state of HIV cure research on display at the Keystone Symposium.

Remission, rebound, reverberation

Timothy Brown is still the only person cured of HIV.

Brown, known as the “Berlin patient,” was cured by two stem cell transplants, both from a donor homozygous for a mutation that knocks out the CCR5 protein receptor from being expressed on cells. This matters because CCR5 is the primary receptor HIV uses to infect CD4+ T cells. The stem cell transplants were part of a panoply of other chemotherapies to treat Brown’s acute myeloid leukemia. In other words, it is hardly a viable cure strategy. Last year Brown’s physician, Gero Hütter of the University of Heidelberg, reported in the New England Journal of Medicine that six other patients from the US, Europe, and South America underwent treatments similar to Brown’s for different types of cancer. None of these patients survived longer than a year. Researchers even wonder if one lesson from these cases is to continue ARV therapy during transplantation instead of stopping it.

Then there was the case of the Mississippi Baby, a newborn who received ARV therapy beginning just 30 hours after birth, even before medical staff had confirmed the baby’s HIV infection status. After a month, researchers could not detect any virus in the infant and therefore stopped ARV therapy. After two years the child remained HIV-free, firing hopes that a cure was achieved. Unfortunately, last summer the child’s virus rebounded and treatment was restarted.

There was also a study involving two HIV-infected individuals known as the “Boston patients” who received stem cell transplants for blood-borne tumors. In this case both patients had heterozygous mutations in their CCR5 genes, but received stem cells from a donor who lacked this mutation entirely. After stopping ARV therapy in the spring of 2013, their HIV levels remained undetectable for months. The virus, however, rebounded by the end of that year.

Deeks, who was one of the first to pick up on the significance of the early research on the Berlin patient, and who himself treated Brown, thinks these cases raise fundamental questions for cure researchers. Where is the viral reservoir located? What is the best way to detect and measure it? And is there a way to activate the reservoir and wipe it out?

For Barré-Sinoussi these questions may be so difficult to answer that a cure may, in the end, turn out to be more akin to a respite. “If you stop treatment, you have viral rebound,” she says. This is because HIV infects cells just before they enter a latent or resting state. In this latent state, they are invisible to the immune system. Active viral replication is efficiently blocked by ARVs, but if therapy is stopped, the latent virus in the reservoir begins actively replicating, resulting in rebound of detectable viral loads. This means that to establish an actual cure, all latently infected cells must be eliminated, Barré-Sinoussi says. “It will be very, very difficult since we have this establishment of the reservoir early on,” she adds. Research in monkeys suggests that the latent reservoir of HIV-infected cells may be established within a matter of days (Nature, doi:10.1038/nature1359). This suggests that while earlier initiation of ARV treatment may help reduce the size of the viral reservoir, it is unlikely to prevent the reservoirs from forming.

“In the case of HIV infection, or in general of retroviral infections which are latent, it is an almost impossible mission to totally eliminate latently-infected cells,” Barré-Sinoussi says. She thinks this is still worth pursuing, but says strategies that allow for a sustained remission from ARV therapy are a more realistic goal. “Remission means to have a persistent reduction and control of the reservoir, without any antiretroviral treatment, and without risk of transmission to others. Remission, in my opinion, is possible.”

But there are still many steps to achieve even this.

The search for a biomarker

When Deeks sees Miles Davenport, leader of the Infection Analytics group at the University of New South Wales in Sydney, he is puzzled and intrigued by the Australian’s new data. Davenport stood in front of a poster showing data from two methods he and his colleagues used to try and measure the frequency of viral rebound following the interruption of ARV treatment among HIV-infected individuals. Davenport aggregated data from four previous human studies of deliberate treatment interruption and used statistical models to analyze data from 100 volunteers. He says that while there have been larger studies, the team examined only those studies that employed regular sampling.

Davenport’s team calculated that it takes an average of five to seven days for HIV to activate from latency following a treatment interruption. They also estimate that viral replication begins on average every six days or so—approximately 24 times more slowly than previously thought. While there may be 100 million actively HIV-infected cells present at any one time in an untreated HIV-infected person, Davenport’s data suggests that a single cell becomes actively infected around once a week when an individual is on suppressive therapy. Prior studies predicted this occurred several times a day. According to Davenport’s calculations, reducing the viral reservoir by 50- to 70-fold, instead of several thousand fold, might therefore be enough to allow for prolonged treatment interruption.

“So do you think time to rebound is a legitimate, gold-standard measurement of the reservoir size?” Deeks asks Davenport. Deeks says he wants to develop a biomarker that will become a surrogate marker for the size of the reservoir during therapy. He isn’t the only one on this quest. If there were a biomarker for reservoir size, researchers wouldn’t have to find ways to measure the reservoir in every patient. This would be a good thing, since they don’t know where the reservoir is entirely anyway. “The gold standard is time to rebound, absolutely,” Davenport says, with Deeks chiming in, “A biomarker that can predict time to rebound: that’s the holy grail of cure research.”

Tissue is the issue

As Mechanisms of HIV Persistence: Implications for a Cure implies, finding, assessing, and understanding the extent of the latent HIV reservoir is paramount to cure research. Deeks says he’s come to believe that relying on blood analysis alone will not accomplish even the first step of this gargantuan task, something he mentions several times during the symposium. “Where exactly is the virus?” he muses. “Where does it actually live during long-term therapy?”

He has one idea. “Many people believe this to be the final frontier, where HIV will actually persist the longest, and that is in the T follicular helper cells within lymph nodes,” Deeks says. T follicular helper (Tfh) cells are a special subset of immune cells found in the follicles of organs in the lymphatic system, such as the spleen and lymph nodes. They play many roles and could be a key component of HIV persistence.

And so tissue, not blood, is where Deeks thinks the hunt for the reservoir should focus. Many of his colleagues agree. “Everyone is looking at blood because it’s easy,” Deeks says. “Most people want to work in tissue, which is great, but it’s not easy. You’ve got to bite the bullet and go in there and do it.” Deeks says there are ethical hurdles to collecting tissue samples from study volunteers. “We have some brave volunteers in the community that are heavily motivated to stimulate cure research, so they participate.”

Olivier Lambotte, an infectious disease expert at the Hôpitaux de Paris who, like Deeks, was a co-organizer of the Boston meeting, and colleagues are studying abdominal subcutaneous and visceral adipose or fatty tissues and their long-term relationship with HIV. Over time, clinicians suspect the body’s fatty tissues change as a result of long-term ARV treatment and the chronic immune activation that results from living with the virus. HIV-infected people can develop lipohypertrophy, which is an unusual fat buildup around the gut. They can also develop lipoatrophy, causing fat to decrease in their legs, arms, and face. Dorsocervical fat pads around the neck and shoulders (sometimes known as buffalo hump) can also accumulate.

Lambotte’s group figures adipose might also provide an ideal environment for HIV persistence, making fatty tissue another component of the viral reservoir. “Tissues are still a black box,” Lambotte says. “We don’t know exactly what happens inside. Most studies have been done in the gut. Other organs are rather badly investigated because it’s difficult to get access to these tissues.” Lambotte says it’s important to include more tissue sample study. “We are on the surface,” he says. “We don’t see what happens in the darkness of the sea. It is a major problem.”

Given the difficulty in obtaining human tissues, Lambotte and colleagues are studying how simian immunodeficiency virus (SIV), the monkey form of HIV, affects the abdominal subcutaneous and visceral adipose tissues in macaques. They found SIV infection increased adipocyte density and caused an enhanced inflammatory profile of adipose tissue immune cells. Lambotte’s group is also working with a small group of HIV-infected volunteers on ARV treatment, analyzing adipose for HIV DNA and RNA. Data from these experiments is pending publication.

Meanwhile, one of Deeks’ colleagues, Joseph Wong, a virologist at the San Francisco Veterans Affairs Medical Center, is showing just how variable, when finally quantified, the reservoir may be in tissues.

Wong found a wide variety of differences in HIV RNA expression levels in biopsies of gut-associated lymphoid tissue and lymph nodes from patients on suppressive ARV treatment. Zian Tseng, a UCSF cardiologist, is conducting a long-term study of sudden cardiac death in HIV patients. Wong was able to piggyback on this study and examine autopsy tissue from eight postmortem individuals who were receiving ARV at the time of death. His team looked at samples from the brain; a series of lymph nodes; the distal ileum, which is the point where small and large intestine intersect; and from the sigmoid colon. Even though it was a limited data set, Wong found a wide range of HIV infection frequency in these different tissues, with as yet no predictable patterns. They found uniformly detectable HIV DNA and RNA in lymphoid tissues, as well as measurable HIV DNA, but not RNA, in brain tissue. The DNA, Wong says, could represent both latent HIV and archived, defective virus. The RNA, however, indicates relatively recent and also possibly persistent viral replication.

Tissue is the issue, Wong says. “It’s not the only issue, but there’s a need to delve deeper into where the virus resides and better understand what are some of the consequences of HIV persistence.” The Keystone audience wanted to know more about the brain tissue, but so far Wong’s only examined one such sample. Not surprisingly these samples are hard to come by. “I know only one volunteer who’s had a brain biopsy,” Deeks says. “That’s Timothy Brown.”

The biochemist Janet Siliciano lights up when she spots Deeks. She and her husband Bob, leading cure researchers from Johns Hopkins University in Baltimore, carry on running debates with the San Franciscan during the symposium week. This time they are debating whether blood can be a true marker for the shadowy reservoir. But the conversation quickly turns to the recent cures that never were—the Mississippi Baby and the Boston patients. She and Bob authored a review about these cases last summer (Science 345, 6200, 1005). “Everyone was getting so discouraged. We wanted to say we’ve learned a lot from these cases, and that’s how we approach it,” Janet says.

What researchers have learned is just how hard this work will be. “These cases give us a really frightening picture of what we’re up against in trying to cure HIV infections,” Bob Siliciano says. “They prove that the virus persists in a latent state for months to years and then begins to replicate.”

Janet and Deeks get back to the blood versus tissue issue. “I am convinced that the blood is not a representative sample of the tissue,” Deeks says. Janet agrees that tissues need analysis. “I talked to Bob this morning,” Deeks replies. “I told him we’ll send you a lymph node tomorrow.”

“Really?” Janet is impressed. “We’re willing to do that,” Deeks says. Even though she thinks studying tissues is important, she’s not convinced that analyzing blood is a bad option. “I firmly believe,” Janet says, “that the blood is representative. Because those cells are circulating throughout the lymphoid system.”

Not T-follicular helper cells, Deeks counters.

“No. They don’t.” Siliciano concedes. “That’s where I agree with you.” And there’s a lot of virus in there, Deeks nudges.

“I’m interested in exploring what’s going on with CD4+ T cells in lymph nodes, and really interested to know if there are any latently-infected T follicular helper cells that survive and return back to a resting state,” Janet says. Deeks, as one of six directors of the 2,000-strong SCOPE cohort of HIV-infected volunteers in San Francisco, takes this recommendation seriously. “We’ll start working on it,” he says.

Flushing out the reservoir

The Siliciano lab is interested in not only characterizing and understanding the viral reservoir, but also eliminating it. One current strategy involves stimulating the reservoir, wherever it is, by shocking the cells in which the virus lies, as Bob and Janet Siliciano write, transcriptionally silent. Once activated they can be killed. This is the basis of the aptly named “shock-and-kill” strategy.

“The dramatic stability of the reservoir is really a major problem that we face,” Bob Siliciano says in Boston. Which is why researchers must take drastic measures to try to rouse the virus from the reservoir and ultimately destroy it. Some shock strategies include using drugs, such as histone deacetylase (HDAC) inhibitors or toll-like receptor (TLR) agonists, to stimulate the reservoir. Some strategies to kill these newly activated cells include using therapeutic vaccines or broadly neutralizing antibodies. Other approaches could include dampening the expression of apoptosis inhibitor molecules, which defend against inflammatory molecules that promote cell death.

Thomas Rasmussen, a researcher in the Department of Infectious Diseases at the Aarhus University in Denmark, is part of a team evaluating different HDAC inhibitors for their potential to activate latent virus. His group is conducting small clinical trials testing panobinostat and romidepsin. One study involves combining romidepsin with a therapeutic vaccine candidate, Vacc-4x, developed by Bionor Pharma. “We would like to combine HIV latency reversal with a vaccine that would increase CD8 responses towards HIV antigens,” says Rasmussen, “to see if that combined approach would augment killing of infected cells stimulated into producing the virus.” He is hoping to present results of this combination study at next year’s Conference on Retroviruses and Opportunistic Infections.

The California biotech Gilead Sciences is dedicating resources to a range of early-stage cure investigations. The company developed a TLR-7 agonist (GS-9620) and is currently conducting dosing studies in early human trials, one of which is currently enrolling HIV-infected volunteers on suppressive ARV treatment (see PrEP Works, IAVI Report, Vol. 19, Issue 1, 2015). Gilead’s director of clinical virology, Romas Geleziunas, says that based on results from monkey studies, it appears this TLR-7 agonist has the potential to both shock latent HIV and kill it. “Some patient cells might be more susceptible to the kick component of this, others the kill. We’re not quite sure,” he says.

Gilead is also experimenting with combining their TLR-7 agonist with the broadly neutralizing monoclonal antibody PGT 121. “We believe the TLR-7 agonist will expose latently infected cells by making them produce HIV proteins,” Geleziunas says. That would include, they think, the surface envelope glycoprotein gp120. If the PGT121 antibody binds to gp120, then it can signal the immune system to kill off the cell. Gileziunas says the company obtained a license from Theraclone Sciences to develop these antibodies and is working on an enhanced version of PGT 121.

A vacation from antiretrovirals

Meanwhile, researchers are mining data from a small but growing cohort of HIV-infected volunteers who have voluntarily stopped ARV therapy and seem to be in a state of prolonged remission. The Institut Pasteur’s Asier Sáez-Cirión and his colleagues first reported on this cohort in 2013. Then there were 14 HIV-infected volunteers in the Visconti cohort, all of whom had started antiretroviral therapy during primary infection and maintained treatment for at least a year before voluntarily deciding to stop treatment. They also all effectively controlled their HIV for at least one year after treatment interruption. In this case control is defined as maintaining a viral load less than 400 copies/ml. Most of the cohort has been in this state for more than a decade now. Sáez-Cirión calls them post-treatment controllers. So far only one Visconti volunteer has rebounded and resumed ARV therapy.

Meanwhile the cohort is growing. Six new volunteers recently joined and the Visconti team is examining 25 other candidate volunteers from outside France and may start enrolling them into the cohort. For Sáez-Cirión, these volunteers suggest that HIV remission is possible. Sáez-Cirión’s team doesn’t yet understand the mechanism responsible for viral control in these individuals. However, the presence of an allele in human leukocyte (HLA) antigen-B is a common thread among the cohort. This HLA-B-35 allele is present in three post-treatment controllers newly identified from Denmark. It is an unexpected observation: this allele is usually associated with high viral loads and rapid progression to disease in the absence of treatment, says Sáez-Cirión. “It’s clearly something you don’t expect in control of infection.”

Sáez-Cirión says results from a study conducted by the Agence Nationale de Recherche sur le Sida convinced him that a small viral reservoir is the likely starting point in creating post-treatment controllers. But if a single HIV-infected cell might be enough to initiate viral rebound, why does the size of the reservoir matter? “If you have one infected cell, the chances of this cell arriving to a place where there are all of the elements for rebound to occur will be extremely rare,” says Sáez-Cirión. His group thinks the fewer infected cells, the easier it is for the host to control.

Going straight to the genes

One bustling evening at Keystone outside the main hall Deeks spots Pam Skinner, a pathobiologist at the University of Minnesota, whom he doesn’t know well. She and Liz Connick, an immunologist at the University of Colorado, are studying the behavior of killer T cells in lymphoid tissue, specifically within lymphoid follicles, where viral replication is concentrated. Her latest data from untreated macaques chronically infected with SIV shows that there are relatively low levels of cytotoxic T lymphocytes (CTL), or killer T cells, in B-cell follicles in lymphoid tissue.

“Here’s the lymphoid follicle. There’s six red cells in there,” Skinner says, pointing to the few bright specks in dark space. “These are the virus-specific CD8+ T cells.” By contrast there was a field of red cells well outside the follicle. “There are too few in here,” she says pointing inside the circle. Skinner speculates that  increasing the number of virus-specific killer T cells in lymph nodes would allow these killer cells to target any latent cells that are hiding out in the lymphoid tissue should they begin actively replicating. Instead of shocking the reservoir inside the body, they’ll use genetic engineering outside of it. 

To test this, Skinner plans to treat SIV-infected monkeys with ARVs until they have undetectable viral loads and then remove their T cells and transduce them with CXCR5; a type of gene therapy. CXCR5 is a chemokine receptor expressed on cells within lymphatic tissues. It directs the migration of B and T cells into lymphoid follicles of the spleen and lymph organs.

Skinner hopes that by transducing the monkey T cells with CXCR5 so that they express this receptor, it will direct them into lymphoid follicles where they could then kill any actively replicating, viral-infected cells. “Ongoing viral replication in the follicles that goes unchecked is the key to HIV/SIV pathogenesis,” says Skinner. “If we can eradicate or better control viral replication in follicles, this may well lead to lower to undetectable viral loads in patients, prevent disease progression to AIDS, and thus, lead to a functional cure.” All without even targeting the latent reservoir.  

Deeks is a little leery of gene-manipulative approaches, calling it an extreme approach. Objections aside, Deeks tells Skinner he’s worked with gene-splicing or gene-therapeutic techniques with some 50 or 60 patients, by his own reckoning. It is just a very challenging field. “You have to get a company to do it,” Deeks says, citing Sangamo, the California biotech he’s worked with in the past. “Gene therapy is not for the weak. You need a lot of resources.”

Paula Cannon, a microbiologist at the University of Southern California, is already working with Sangamo. She is now preparing for early-phase clinical trials testing a gene therapy strategy intended to knock out the same CCR5 co-receptor gene that produced such an astounding result in Timothy Brown. The strategy is to remove hematopoietic stem cells from an HIV-infected volunteer and treat the cells outside the body with a zinc finger nuclease using messenger RNA as a vector. The treated cells are then delivered back into the volunteer after a mild dose of chemotherapy to promote re-engraftment of the engineered cells. While this is similar to trials run four years ago at the University of Pennsylvania, Cannon’s team will be treating stem cells instead of T cells. Their thinking is that those stem cells will give rise to T cells in the body and be a longer-lasting treatment than engineering T cells.

Cannon is also working with an adeno-associated virus vector in combination with the zinc finger nuclease messenger RNA. “It allows us to not only knock out a gene, but make precise edits to that gene,” she says. It’s an approach that’s not yet ready for clinical testing, but may make it possible to make additional genetic changes that could confer HIV resistance.

This conversation will continue when cure researchers meet in Vancouver in July for the 2015 Towards an HIV Cure Symposium. After Boston, it seems that remission may be the first stop on the road toward a cure. “It’s an important point to show that remission, even if it is long-term remission, cannot be definitive,” Sáez-Cirión says. “We have one patient who is very clearly losing control of infection even after many years. Remission is remission. It does not imply that it is a cure.”

Michael Dumiak reports on global science, technology, and public health and is based in Berlin.