Shocking HIV Out of Hiding: How Are We Doing?

The key obstacle to an HIV cure remains the reservoir of persistent virus, which is impervious to standard anti-HIV drugs. It has been a target of numerous research teams, many of which are focused on a “shock and kill” approach: dormant HIV-infected cells are activated and thus rendered susceptible to drug- and immune-based attack.

Dr. Sharon Lewin

However, although a few trials of latency-reversing agents (LRAs) in HIV-positive patients on antiretroviral therapy (ART) have shown the expected increase in cell-associated HIV genetic material in T cells, the frequency of those patients’ latently infected cells hasn’t budged. Writing in the July issue of Current Opinion in HIV and AIDS, amfAR-funded scientist Dr. Sharon Lewin of the University of Melbourne, along with Dr. Thomas Rasmussen from Aarhus University Hospital in Denmark, suggests three reasons for this persistent failure in the clinic.

First, such a strategy demands a robust shock to the latent state. More effective LRAs, possibly used in cocktails similar to combination ART, are required. Second, immune responses that are potent enough to kill those induced virus-expressing cells are needed. Finally, Drs. Lewin and Rasmussen believe more attention should be paid to the role of immune escape mutations among newly awakened virus, i.e., viruses that may evade attempts to destroy them using vaccine strategies or antibodies.

Dr. Warner Greene

amfAR is funding several groups to examine those issues, in the test tube and in clinical trials. A recent proof of concept for this approach was provided by amfAR-funded scientist Dr. Warner Greene and colleagues at the University of California, San Francisco. Writing in the prestigious journal Nature Medicine, they recognized that certain types of retinoic acid—vitamin A-based derivatives—can activate genes that increase HIV growth as well as preferentially induce the death of HIV-infected cells.

One of these derivatives is acitretin, an FDA-approved pill for the treatment of psoriasis. Dr. Greene and associates discovered that acitretin was particularly effective in decreasing proviral DNA—a sensitive marker for latency—in CD4+ T cells of HIV-positive individuals on ART, particularly when used together with a common LRA known as SAHA.

At the moment these are promising test-tube experiments. But the authors conclude that their model might be the improved “shock and kill” strategy needed to eliminate all HIV-infected cells.

Dr. Laurence is amfAR’s senior scientific consultant.

Antibodies as an Important Part of an HIV Cure Strategy

Last month, we reviewed the work of amfAR-funded scientist Dr. Nancy Haigwood of Oregon Health and Science University, who is exploring how to block mother-to-child transmission of HIV. She used a monkey model and a “passive immunotherapy” strategy based on a cocktail of two potent antibodies capable of neutralizing a broad spectrum of AIDS viruses. In the April issue of Nature Medicine, she and her colleagues wrote, “early passive immunotherapy can eliminate early viral foci and thereby prevent the establishment of viral reservoirs.” And anything that can affect HIV reservoirs is of strong interest to cure researchers.

This point is now being aggressively pursued by amfAR Krim Fellow Dr. Stylianos Bournazos and associates working in the laboratory of Dr. Jeffrey Ravetch at The Rockefeller University in New York. Writing in a May issue of the prestigious journal Science, the researchers reported utilizing a single broadly neutralizing anti-HIV antibody to target infected CD4+ T cells in mice with a humanized immune system (i.e., mice that have been injected with human stem cells). They found that the survival of infected cells could be greatly decreased by this antibody through a process involving an immune receptor known as Fc gamma.

This work is important as antibodies differ from anti-HIV drugs in that they can alter the survival times of both cell-free virus and infected cells. They can also recruit host immune cells to defend against the virus. A variety of different strains of HIV obtained directly from patients were used in this mouse model, and the antibody was equally effective against all of them. In addition, another mouse model mimicking chronic HIV infection demonstrated that such antibodies can accelerate clearance of cells after a longer-term HIV infection.

The authors concluded with this promising statement: “The finding that antibodies can clear infected cells in vivo has important implications for therapies aimed at HIV prevention and viral reservoir reduction or elimination.”

Dr. Laurence is amfAR’s senior scientific consultant.

Exposing HIV-Infected Cells to Antibody Attack

Exposing HIV-Infected Cells to Antibody Attack

An HIV-infected T cell (Source: NIAID)

Interest in the role of antibodies—traditionally thought of as a vital component of any vaccine designed to prevent HIV infection—has been growing in the context of HIV cure research. This was reflected in a meeting— Development of Monoclonal Antibodies for HIV Treatment and Cure—convened by the National Institutes of Health and the Gates Foundation, June 3–4, in Rockville, MD.

Antibodies are generally thought of for their ability to neutralize virus that has not yet infected cells, whereas the main stumbling block in curing HIV is cells that are already infected. But antibodies have additional functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC), that can directly target infected cells. ADCC occurs when a cell of the immune system—an effector cell such as a natural killer (NK) cell—binds to an antibody that in turn binds to the surface of an infected cell. The NK cell can then kill the infected cell.

Writing in the May issue of the Proceedings of the National Academy of Sciences, amfAR fellow Dr. Navid Madani of Harvard Medical School and a large team of researchers described a method of boosting the ability of antibodies to kill HIV-infected cells via ADCC. The ADCC effect hinges on the presence of the Env protein on the surface of infected cells. Env originates on the surface of the virus and binds to CD4 proteins on susceptible cells. After infection, some Env remains on the cell surface.

HIV protects itself from antibody attack by using two of its own proteins, Nef and Vpu, to decrease exposure of Env on the cell surface. To override this mechanism, Madani and colleagues used various forms of infecting virus, as well as variations of the CD4 protein, to enhance the ADCC-mediated killing of infected cells generated in a petri dish, as well as in cells taken from HIV-positive individuals. The authors envision that the combination required for this approach might be delivered by vaccine (for the antibodies) plus an oral formulation of modified CD4 proteins.

Dr. Madani collaborated with researchers from the University of Montreal; University of Pennsylvania; Institut de biologie et de technologie de Saclay, France; University of Maryland; Johns Hopkins University; the Scripps Research Institute; and Duke University.

amfAR recently funded another project that will combine the antibody 3BNC117—optimized for ADCC—with the cancer drug romidepsin, the most promising agent to date in terms of its ability to shock the virus out of latency. For more details, click here.

Dr. Johnston is amfAR’s vice president and director of research.

Attacking HIV in the Gut

Dr. Mirko Paiardini

Most research approaching an HIV cure has centered on T cells in the blood. This is understandable, given ready access to such samples. But it is now apparent that, both in HIV-infected humans and in SIV-infected monkey models, one of the very first immunologic events early after infection is severe depletion in the intestines of certain specialized T cells, Th17 and Th22. These cells are recognized by their production of two immune hormones, the interleukins IL-17 and IL-22, respectively. Writing in the February issue of the journal PLoS Pathogens, amfAR-funded scientist Dr. Mirko Paiardini of Emory University, together with colleagues there and at the University of Montreal, report on the function of these cells and the ability of antiretroviral therapy to maintain the cells’ function after infection.

The researchers found that these cell types in healthy monkeys and humans produced at least five critical cytokines necessary to maintain integrity of the intestinal lining. The level of their depletion after an SIV infection in their experimental monkey model directly correlated with levels of immune activation, established early after an infection. Following treatment with ART, improvement in terms of Th17 T-cell number and function inversely correlated with levels of virus in the animal’s blood. In other words, the higher the level of virus, the less recovery there was in the function of IL-17-producing cells. On the flipside, more of those intact cells predicted less of the virus.

Of great significance for studies of potential HIV cures, it was found that after stopping ART in the infected monkeys—as would be done as part of an analytic treatment interruption, or ATI (to help establish that an individual with no detectable virus after an experimental cure trial was truly virus-free)—both Th17 and Th22 T cells were rapidly and severely damaged.

The authors concluded that it was important to preserve intestinal T17 and T22 T-cell function during HIV infection. They emphasized the need for “therapeutic strategies aimed at improving these cells’ function in future HIV cure research.”

CTLs: Stormtroopers of the Immune System

An award-winning image of CTLs attacking a tumor cell (Photo: Sudha Kumari)

Dr. Jones is the recipient the first amfAR grant to be funded by generationCURE, a group of young professionals dedicated to advancing amfAR’s efforts to find a cure for HIV. A major obstacle to HIV eradication is the presence of a reservoir of virus that is established soon after infection and lies dormant during effective antiretroviral therapy. Eradicating the cells that harbor that virus is the goal of research aimed at a sterilizing cure (in which all virus is completely eliminated from the body). One approach being taken by researchers to accomplish such a cure is to recruit the help of a subset of immune cells charged with clearing the reservoir.

Natural killer (NK) cells and cytotoxic T cells (CTLs) mediate the killing of virally infected cells and, in the case of CTLs, can be specific for HIV.  Much research has been devoted to retraining CTLs to be better killers of HIV-infected cells. Writing in the February issue of The Journal of Clinical Investigation, Drs. Brad Jones and Bruce Walker review the properties of CTLs that could be exploited as part of a cure for HIV.

Dr. Jones is Assistant Professor in the Department of Microbiology, Immunology and Tropical Medicine at The George Washington University. He gained expertise in CTLs during his postdoctoral work in the lab of Bruce Walker, who was one of the first to describe CTLs in HIV-infected persons. Dr. Jones has published numerous studies on CTLs and continues to pursue his research with a grant from amfAR’s generationCURE. Here, he shares his views on the latest approaches being used by scientist to harness CTLs in the search for a cure for HIV.  

You recently published a review article with Dr. Bruce Walker on the role of CTLs in HIV cure studies. What makes this topic especially timely?

Dr. Jones is the recipient of the first amfAR grant to be funded by generationCURE, a group of young professionals dedicated to advancing amfAR’s efforts to find a cure for HIV.

CTLs, also known as CD8+ T cells, are cells of the immune system that specialize in recognizing and killing virus-infected cells. I always find it inspiring to see these guys in action—moving quickly through tissues and hunting for virus-infected cells. When a CTL encounters an infected cell it rapidly discharges toxic proteins, killing the target cell, essentially eliminating a virus factory. The sensitivity and specificity of this killing is really impressive.

Even more impressive is the impact CTLs can have in a clinical setting. Louis Picker, Jonah Sacha, and colleagues in Oregon, for example, have developed a means of eliciting a CTL response that is capable of completely eradicating SIV (the monkey version of HIV) from animals. The CTLs elicited in this study have a very unusual way of recognizing infected cells, but serve to illustrate the power of these types of cells if they can be properly directed.

“I always find it inspiring to see these guys in action—moving quickly through tissues and hunting for virus-infected cells.”

The challenges involved in either completely eradicating HIV infection (sterilizing cure) or enabling the immune system to control infection long-term (functional cure) are both considerable. In my opinion, our best chance at achieving these goals centers on finding ways to aim and enhance the natural antiviral weapons in our immune system, such as CTLs, against persistent virus. 

Why do CTLs fail to clear HIV infection? 

There are several factors at play here. The classical explanation would be that CTLs cannot recognize the viral reservoir because it hides in a latent state. This is certainly part of the answer, but I would speculate that there is some ongoing interaction between CTLs and the viral reservoir, even in individuals with undetectable viral loads.

Another part of the explanation is that HIV has an uncanny ability to mutate in order to ‘escape’ detection by CTLs. We also know that CTLs become ‘exhausted’ when faced with persistent infections such as HIV and less effective at eliminating infected cells. However, there is still a lot that we don’t know. Several labs, including my own, are interested in determining what makes a CTL particularly effective against the viral reservoir so that we can hopefully elicit these responses therapeutically.

What are some of the approaches scientists are using to make CTLs more effective?

I would highlight two studies that I am involved in. In one project funded by amfAR’s generationCURE, we are studying a drug that has the potential to act as both an immunomodulation agent and a latency-reversing agent – thus simultaneously exposing infected cells to CTLs and enhancing the ability of CTLs to kill these targets. In a second project we are exploring immunomodulation using an antibody that enhances the activity of IL-21, a molecule that improves the killing ability of CTLs.

Definitions:
Immunomodulation: The use of drugs or biologics (antibodies, cytokines, etc.) to alter the immune response.
Latency reversal: Latency-reversing agents wake up dormant HIV and facilitate elimination of infected cells.

Dr. Michel Nussenzweig is working with colleagues to test broadly neutralizing antibodies in a combination cure approach in an amfAR-supported clinical trial.

Dr. Michel Nussenzweig is working with colleagues to test broadly neutralizing antibodies in a combination cure approach in an amfAR-supported clinical trial.Are there any CTL-focused trials currently in the clinic?

Yes, there are quite a few trials of therapeutic HIV vaccines aimed at enhancing CTL responses, including some that combine vaccination with latency-reversing agents to try to ‘shock and kill’ the reservoir. Cath Bollard, in collaboration with David Margolis, is also in the process of testing CTL therapy – whereby HIV-targeting CTLs are isolated from participants, expanded and then re-infused, with the hope that these will drive reductions in the reservoir. I’m anxiously awaiting the results from these studies. If we can see a signal that the reservoir is significantly reduced in some individuals, then we will have something that we can grab onto and further improve upon – this is really the breakthrough that we are all working towards.

What strides have been made in HIV cure research that make you hopeful that we will find a cure, sterilizing or functional, in the near future?

In regard to a sterilizing cure, I would again point to the work of Louis Picker, Scott Hansen, Jonah Sacha and others at Oregon Health Sciences University with their demonstration that an unusual type of T cell response is able to eradicate SIV infection from monkeys. There are some key limitations of the current form of this intervention – first, the vaccine has thus far been given before an animal is infected and, second, it is only effective in approximately 50% of animals. However, to my knowledge, this is the first and only example of immune-mediated elimination of infection with a lentivirus.

“I commend amfAR for being highly involved in pushing this research forward.”

Stepping away from CTLs for a moment, I am also encouraged by the work being performed with broadly neutralizing antibodies as reported by Michel Nussenzweig, James Whitney, Dan Barouch, and others. We have an incredible arsenal of very effective antibodies available and evidence is mounting that these can be used to target the viral reservoir. I commend amfAR for being highly involved in pushing this research forward.

Gilead has presented exciting data showing that a small-molecule TLR-7 agonist is able both to reverse HIV latency and to enhance the functions of both CTLs and NK cells, representing a potentially powerful combination.

In relation to a functional cure, the phenomenon of post-treatment control of HIV for individuals who started treatment early in infection, best represented by the VISCONTI cohort in France, provides an important precedent that long-term immune control is possible. If the mechanisms underlying this control can be determined, then we will have a path forward to attempt to induce the appropriate responses in a broader array of individuals.

There is really a tremendous amount to be excited about in this rapidly moving field and we are extremely fortunate to be working with a community that is supportive of our efforts, both in terms of financial support and by volunteering to participate in clinical studies. As long as we continue to pull together, we will achieve the goal of curing HIV – either through one of the approaches highlighted above or, perhaps, through something entirely unexpected.

Are Early Immune Defenses Helping HIV Flourish?

A plasmacytoid dendritic cell (blue) interacting with a T-cell (pink).

Our immune systems offer a very rapid, non-specific first line of defense against invading viruses. These innate pathways lack the specificity and memory characteristic of our more mature immune defenses, which can take weeks to evolve, but they are essential in the control of many infectious diseases. Writing in the June issue of The Journal of Infectious Diseases, amfAR-funded scientist Dr. R. Keith Reeves and colleagues from Harvard Medical School and the New England Primate Research Center dissect the role of one of these early defenses in the control of HIV.

Using a monkey model for AIDS—SIV infection in the macaque—Reeves and associates examined CD4+ plasmacytoid dendritic cells (pDCs), a potent source of interferon-alpha. By bringing interferon to sites of viral invasion and growth, pDCs can inactivate many viruses before they have a chance to replicate and spread. They can also amplify the killing effects of another form of innate immunity against viruses, natural killer cells. The importance of pDCs is emphasized by the fact that they are present at high levels in elite controllers of HIV infection, but are depleted in most humans, and monkeys, soon after infection occurs. They rise again after the introduction of antiretroviral therapy.

But how much of a trade-off does this innate, interferon-based defense present in the context of HIV? It is a key concern because too much interferon leads to inflammation in the gut and other tissues. As regular readers of these updates will recognize, inflammation contributes to HIV growth, and to premature aging of the heart, kidney, and bones in HIV-positive individuals.

Reeves and colleagues discovered that the production of interferon by pDCs occurred too late to control acute SIV infection. Additionally, pDCs failed to migrate to and accumulate in the gut—a major source of early SIV and HIV growth—until after virus levels were well established in the blood. And they may also be pathologic, contributing to ongoing immune activation. Whether this system should be modulated as part of strategies to enhance the control, and eventual cure, of HIV requires further investigation.

Dr. Laurence is amfAR’s senior scientific consultant.

The Science, and Ethics, of Stopping ART in Assessing Possible HIV Cures

A variety of different approaches to HIV eradication have been discussed in these updates. But this update is a bit different, describing a critical issue: How might we better assess the likelihood of HIV resurgence after stopping ART and, if possible, the probable time frame for such a recurrence, in a given patient?

Dr. Timothy Henrich

Writing in the January issue of The Lancet HIV, amfAR-funded scientists Timothy Henrich of the University of California, San Francisco (UCSF), and Sharon Lewin of the University of Melbourne, with colleagues from Johns Hopkins University and UCSF, note that a stem cell transplant approach that replicates the experience of the “Berlin patient” remains “the only approach that has led to a meaningful decrease in latently infected cells in individuals on ART."

The Berlin patient is a formerly HIV-positive man with leukemia, who in 2008 became the first and only person known to have been cured of HIV after he received a stem cell transplant from a donor with a rare genetic mutation (the CCR5 mutation) that confers resistance to HIV infection. The authors state that such a procedure remains “the only approach that has led to a meaningful decrease in latently infected cells in individuals on ART.” The most definitive way to document another such cure, as a result of stem cell transplantation or any other intervention, requires that the person be taken off anti-HIV drugs.

Dr. Sharon Lewin

But this so-called analytical treatment interruption has risks, which raise several ethical questions. Particularly in the realm of stem cell transplantation, aggressive viral rebound has been seen, with associated morbidity, possible mortality, and a chance of the emergence of a drug-resistant virus. Risks to sexual partners in case of such rebound must also be recognized. The need for very close, very frequent monitoring with available technologies to assess viral load and latent virus reservoirs is cumbersome, costly, and may involve years of surveillance.

But, as Henrich and associates note, “An understanding of the mechanisms by which [these transplants] can result in sustained ART-free remission or the reasons why it fails to do so could reveal key information to advance HIV cure research.”

With these concerns in mind, amfAR is hosting a think tank in Memphis in early March to try to identify methods by which HIV relapse can be predicted following a treatment interruption related to a cure trial. We’ll keep you informed.

Breast Milk Yields Clues to Natural Inhibitors of HIV

Breast Milk Yields Clues to Natural Inhibitors of HIV

Dr. Angela Wahl

Most infants breastfed by an HIV-positive woman do not become infected. It has long been known that components of human breast milk from HIV-negative mothers can inhibit the growth of HIV in the test tube, indicating that HIV-specific antibodies in milk are not responsible for this activity. But until Dr. Angela Wahl, an amfAR Krim Fellow, began her studies, no one had explored such a phenomenon in HIV-positive women.

Writing in the November 2015 issue of the Journal of Virology, Dr. Wahl, working at the University of North Carolina in Chapel Hill, along with colleagues from Duke, Harvard, Columbia, UC-San Diego, and the University of Southern California, explored the activity of milk derived from HIV-positive women from Zambia who had breastfed their infants and either transmitted the virus or not. Rather than limiting experiments to test-tube studies and oral transmission, Dr. Wahl used “humanized mice” carrying components of a human immune system, and explored vaginal and intravenous HIV transmission as well.

All humanized mice exposed orally, vaginally or intravenously to small doses of HIV became infected. In contrast, only a quarter of the animals became infected when the virus was introduced orally along with human breast milk. It was 25% regardless of whether the milk came from known virus transmitters, or those Zambian women who breastfed and did not transmit. Significant protection was also obtained using virus administered with breast milk by the vaginal and intravenous routes.

The nature of the active components in human breast milk that are responsible for this protective effect remains unclear. The activity was not found in milk from cow, camel, goat, or monkey. It was not destroyed by pasteurization or by manipulations designed to remove or destroy protein, sugars, or fat. The authors concluded that the activity most likely involved an interaction among those three components.

Given the nutritional and immunologic benefits of breast milk, and the dangers of preparing infant formula when clean water cannot be guaranteed, these data bolster current recommendations that HIV-positive women in resource-poor nations exclusively breastfeed in combination with antiretroviral therapy. The promise is that identification of the components responsible for the anti-HIV activity might form the basis for the development of natural microbicides—substances that could be applied topically to prevent acquisition of HIV infection—or other anti-HIV medications.

Dr. Laurence is amfAR’s senior scientific consultant.