© JUSTIN GABBARD
Since the early 1980s, when HIV was first identified, our knowledge of the virus—how it causes disease, how it interacts with our immune system, how it responds to drugs—has grown year by year. Drugs specifically designed to target HIV, and given as a cocktail of different agents—known as combination antiretroviral therapy (ART)—have decreased the mortality associated with HIV infection to the point where, for newly diagnosed individuals today, life expectancies are comparable to those who are HIV-negative.
But of the 35 million people currently living with HIV, the World Health Organization estimates that only around 40 percent use ART, partly because about half do not know they are infected. Providing ART to all who need it is a major challenge, and even when the drugs are available they are not a panacea. Regardless of treatment, there is increasing evidence that HIV-infected individuals may be at greater risk of non-AIDS comorbidities, for example, cardiovascular disease and dementia. Moreover, ART has to be taken for life: if the drugs are stopped, virus production quickly ramps up and the disease can progress, a phenomenon known as rebound.
Rebound occurs because HIV forms a reservoir in long-lived T cells that persists despite treatment. As with all retroviruses, a key aspect of the HIV replication cycle is the reverse transcription of the viral genome into DNA, followed by integration of this viral DNA, known as the provirus, into the host genome. (See illustration.) In activated cells, this proviral DNA can give rise to viral mRNA, proteins, and infectious viral particles. However, in some infected cells, the virus enters a resting state, termed latent infection, in which transcription or translation is restricted but integrated HIV is still present. These latently infected cells make up the HIV reservoir and, eventually, may be stimulated to produce infectious virus.
One challenge in targeting latently infected cells is that they do not produce HIV antigens and are therefore indistinguishable from uninfected cells.
The HIV reservoir consists largely of resting CD4+ T cells, but other cells, such as macrophages, may also contribute. In patients who have been treated for many years with ART, these latently infected cells are rare, but still present. It has been estimated that the proportion of latently infected cells capable of giving rise to rebound virus production is approximately one in a million resting CD4+ T cells in patients on ART.1 However, the difficulty of reliably measuring the reservoir means this number could be significantly higher or lower. (See “Ethical Dilemmas.”) Regardless, the HIV reservoir is a major barrier to virus eradication, and its existence raises several questions for cure strategies. Is it possible to completely eradicate latently infected cells from the body, or can we keep them silent to prevent viral rebound? Even more to the point, is it possible to prevent the reservoir from forming in the first place?
Different types of cure
© EVAN OTO/SCIENCE SOURCEDue to the assimilation of viral genetic elements into the host genome, researchers previously assumed that, once infection has taken hold, HIV can never be completely eliminated from the body. Yet in 2009, German clinicians announced the case of an apparent HIV cure in Timothy Ray Brown, also known as “the Berlin patient.”2 Brown underwent a bone marrow transplant following unsuccessful treatment for acute myeloid leukemia with conventional chemotherapy. The bone marrow donor selected by Brown’s clinicians was homozygous for a mutation in the CCR5 gene, preventing the expression of the CCR5 HIV coreceptor on the surface of T cells and conferring a high degree of natural resistance to HIV infection. Following the transplant, Brown ceased taking ART, and the virus did not rebound. More than six years later, researchers have been unable to find evidence of replication-competent HIV in blood or tissues from this patient; it appears that any viral reservoir has been cleared.3 Despite the exceptional circumstances surrounding this case, many believe that the Berlin patient serves as proof of concept that HIV can be cured.
Researchers have since attempted bone marrow transplants from donors carrying the same CCR5 mutation in six other cases of HIV-positive patients. Unfortunately, all of these individuals died within a year from relapsed malignancy or transplantation complications.4 In one of these individuals, rebound occurred after an HIV variant used an alternative T-cell coreceptor, CXCR4, suggesting a potential limitation to targeting only CCR5.5
In addition to preventing the spread of the virus to new cells within body, efforts to “cure” HIV have focused on reducing the size of the reservoir to achieve a remission or functional cure, in which patients could remain off therapy without rebounding, despite detectable HIV DNA in their bodies.
These cases demonstrate the intrinsic dangers and difficulties of the Berlin patient strategy, which could never be realistically scaled up to help all those infected with HIV. Interestingly, when two patients in Boston underwent bone marrow transplants using tissue from a donor carrying wild-type CCR5, their viral levels dropped to undetectable levels, both in plasma and intracellularly, and these low levels endured for several years.6 Unfortunately, viral rebound occurred within a few months of stopping ART, indicating that the CCR5 mutation was indeed critical to the Berlin patient’s cure.7 This has led researchers to another strategy to eradicate HIV: using gene therapy to turn off CCR5 expression. If successful, such a treatment could prevent additional cells from being infected with HIV, thwarting disease progression even in the presence of a viral reservoir. (See “Genome Editing Cuts Out HIV,” The Scientist, July 21, 2014.)
Early treatment, better control
While researchers have traditionally envisaged a “sterilizing cure,” in which the virus is completely eliminated from the body, this may not be necessary for controlling the infection. In addition to preventing the spread of the virus to new cells within body, efforts to “cure” HIV have focused on reducing the size of the reservoir to achieve a remission or functional cure, in which patients could remain off therapy without rebounding, despite detectable HIV DNA in their bodies.
Serving as an example of functional cure, 14 patients across France known as the VISCONTI cohort have successfully stopped ART without return of virus production, some for many years. Unlike the Berlin patient, these patients still have detectable, although small, HIV reservoirs that could serve as a future source of viral reactivation.8 Nevertheless, the fact that these patients have achieved long-term posttreatment control (PTC) could provide important clues for the development of an HIV cure.
A key factor with the VISCONTI cohort is that these individuals started treatment very soon after being infected. Although not all patients treated early are able to cease ART without rebound, several other cohorts that commenced ART during early stages of infection have also achieved varying levels of PTC. While viral rebound occurs rapidly in most individuals, between 5 percent and 15 percent of patients remain free of virus at 24 months after ART cessation.8,9 In contrast, PTC is rarely, if ever, observed among patients who commenced ART only after their CD4+ T cell counts declined below a cut-off point, suggesting that starting treatment during primary HIV infection appears to be important for controlling the virus. Further evidence for the potential of this strategy comes from the reported cure of an infant in Mississippi who, perinatally infected with HIV, commenced ART 30 hours after birth.10 Treatment was discontinued at 18 months of life, and HIV levels remained undetectable for more than a year, until rebound occurred in July 2014.11
The SPARTAC (Short Pulse Antiretroviral Therapy at HIV Seroconversion) trial put early ART to the test in a large randomized trial that ran from 2003 to 2010 across eight countries. Adults testing positive for HIV received a short course of ART within 24 weeks of seroconversion, when an HIV-specific antibody becomes detectable in the blood, resulting in delayed CD4+ T cell decline. The trial also showed that levels of HIV DNA correlated with this delay and predicted time to viral rebound, a finding that links early treatment with reduced reservoir size, possibly as a result of limiting the initial seeding of the reservoir.12,13 Because it is almost certain that a cure will be easier to achieve in patients with lower numbers of latently infected cells, it seems that early treatment will be an important part of HIV eradication strategies.
As most patients are not diagnosed with acute HIV infection and are unable to initiate ART early on, treatments are also needed to deplete the HIV reservoir once it is established. One challenge in targeting latently infected cells is that they do not produce HIV antigens and are therefore indistinguishable from uninfected cells. This transcriptional silence renders these cells invisible to immune surveillance and allows persistence of provirus over time. Although counterintuitive, waking the virus from its latent state may be the key to eradicating the reservoir, by rendering reservoir cells susceptible to immune clearance and other targeted treatments. Researchers are developing strategies combining drugs to reactivate latent virus and techniques to boost immune clearance of infected cells—a so-called kick-and-kill approach.
Transcription of proviral HIV DNA is dependent on the recruitment of appropriate transcription factors to the viral 5’ long-terminal repeat (5’ LTR). As with all genes, chromatin arrangement around the site of viral integration is an important regulator of transcriptional status. High histone acetylation at the 5’ LTR is associated with an accessible chromatin structure, favoring transcription. Acetylation status is maintained by a balance between histone acetyltransferases, which act to promote acetylation, and histone deacetylases (HDACs), which decrease acetylation. HDAC inhibitors are drugs that promote nonspecific acetylation and activate cells latently infected with HIV both in vitro and in vivo.
Vorinostat is an HDAC inhibitor used to treat cutaneous T-cell lymphoma in humans. It has been shown to disrupt HIV latency in cellular models and in primary CD4+ T cells from HIV-infected patients. Trial doses of vorinostat given to HIV-infected individuals with ART-suppressed viral replication caused increases in cellular acetylation and subsequent HIV transcription, but with no evidence of rebound.14,15 In other words, the latent cell was activated enough to start the virus life cycle, but no new virions were detected. This could be due to posttranscriptional barriers to viral expression, such as mRNA degradation, or suboptimal dosing regimens. Although it is unclear what level of expression is needed to trigger immune clearance, it is presumed that at least protein expression, if not assembled virus, will be needed. Researchers at Aarhus University in Denmark have shown that two other HDAC inhibitors, panobinostat and romidepsin, have higher potency and can induce virus production in patients treated with ART.16,17 Despite reactivation of latent viruses, however, neither of these drugs resulted in decreases in reservoir size. But given the short time period of these studies and the small number of patients, this is not altogether unexpected, and the results serve as evidence that it is possible to reactivate the reservoir in vivo.
Although counterintuitive, waking the virus from its latent state may be the key to eradicating the reservoir, by rendering reservoir cells susceptible to immune clearance and other targeted treatments.
Although HDAC inhibitors are the most widely studied class of HIV-activating drugs, several other candidates may also be able to reverse HIV latency. These include methyltransferase inhibitors, protein kinase C agonists (prostratin and bryostatin), the BET bromodomain-inhibiting molecule JQ1, and the zinc-chelating agent disulfiram. The distinct mechanisms of these agents mean that toxicities and efficacy will differ, and it is unclear which agents will be most suited for clinical use. Human trials using HDAC inhibitors and these other HIV-activating agents are exploring the effect of these drugs on viral transcription and translation, as well as the drugs’ safety and tolerability.
Even if HIV-activating agents can provide the kick needed to disrupt HIV latency, there is no evidence that this will result in clearance of the reservoir by the immune system alone. As such, it is likely that these drugs will need to be used in combination with other strategies to promote immune clearance of these cells. The RIVER (Research in Viral Eradication of HIV Reservoirs) trial, for example, is combining vorinostat with vaccination in patients on ART in the U.K. A similar study in Denmark using the HDAC inhibitor romidepsin, and a different vaccine candidate is also underway.
Putting cure into context
Significant work remains to be done in the development of a potential cure for HIV. It is an exciting time in the field, with interventional trials running in parallel with continued basic research into the mechanisms of HIV infection and pathology. In addition, a number of large observational studies of the dynamics of reservoir size are underway to understand PTC, which may provide insights into factors that predict its occurrence, the mechanisms of reservoir formation and maintenance, and the probability of rebound.
Meanwhile, several different therapeutic approaches to an HIV cure are currently under investigation. In addition to gene therapy attempting to damage the CCR5 gene or block its expression, researchers are developing therapeutic HIV vaccines. (See “Defeating the Virus.”) Antibodies with broad HIV-neutralizing capability may have some in vivo activity against the virus, and transfusions of these are being trialled. Coupled with HIV-activating drugs, these immune augmentation strategies are important early steps towards a cure.
Of course, research into an HIV cure is only one of a multitude of approaches to solving the challenges posed by this virus. Prevention, testing, treatment, and managing comorbidities remain the cornerstone of HIV management and research worldwide. Curative interventions, while exciting, are still in very early stages of development; it is unlikely that these are going to play an important role in treatment of infected patients in the near future. Despite this, the prospect of an HIV cure remains a driving force for many, and a successful cure must, in some way, address the reservoir. In this rapidly progressing field, the next few years will be crucial in determining whether clearance of the reservoir may one day be possible.
Genevieve Martin is a doctoral student in the Nuffield Department of Medicine at the University of Oxford, U.K. Matthew Pace is a postdoctoral researcher in the same department, as well as a James Martin Research Fellow at the Institute for Emerging Infections in the Oxford Martin School. John Frater holds positions at both institutions, as well as at the Oxford National Institute of Health Research Biomedical Research Centre, and is a clinician at the John Radcliffe Hospital in Oxford.
In the absence of robust methods for detecting latently infected cells, treatment interruption remains the sole way of testing HIV cure interventions. Ceasing therapy, however, risks resumption of HIV production and reseeding of the reservoir, which may impact disease progression and allows for transmission of the virus.
Evidence of adverse outcomes after treatment interruption comes from the SMART study, in which such interruption was associated with increased mortality, opportunistic infections, and major non-AIDS comorbidities in chronically infected patients (N Engl J Med, 355:2283-96, 2006). Importantly, the SMART study left patients untreated for long periods of time despite high levels of viremia. Researchers have not reported adverse outcomes in studies where patients were immediately placed back on therapy after viral rebound. Nevertheless, these findings highlight the need to consider both the risks and the ethical implications of this strategy. Close monitoring of patients during treatment interruption with clear plans for restarting ART need to be part of cure intervention trials.
- D. Finzi et al., “Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy,” Nat Med, 5:512-17, 1999.
- G. Hütter et al., “Long-term control of HIV by CCR5 Delta32/Delta32 stem-cell transplantation,” N Engl J Med, 360:692-98, 2009.
- S.A. Yukl et al., “Challenges in detecting HIV persistence during potentially curative interventions: A study of the Berlin patient,” PLOS Pathog, 9:e1003347, 2013.
- G. Hütter et al., “More on shift of HIV tropism in stem-cell transplantation with CCR5 delta32/delta32 mutation,” N Engl J Med, 371:2437-38, 2014.
- L. Kordelas et al., “Shift of HIV tropism in stem-cell transplantation with CCR5 Delta32 mutation,” N Engl J Med, 371:880-82, 2014.
- T.J. Henrich et al., “Long-term reduction in peripheral blood HIV type 1 reservoirs following reduced-intensity conditioning allogeneic stem cell transplantation,” J Infect Dis, 207:1694-702, 2013.
- T.J. Henrich et al., “Antiretroviral-free HIV-1 remission and viral rebound after allogeneic stem cell transplantation: report of 2 cases,” Ann Intern Med, 161:319-27, 2014.
- L. Hocqueloux et al., “Long-term immunovirologic control following antiretroviral therapy interruption in patients treated at the time of primary HIV-1 infection,” AIDS, 24:1598-601, 2010.
- S. Lodi et al., “Immunovirologic control 24 months after interruption of antiretroviral therapy initiated close to HIV seroconversion,” Arch Intern Med, 172:1252-55, 2012.
- D. Persaud et al., “Absence of detectable HIV-1 viremia after treatment cessation in an infant,” N Engl J Med, 369:1828-35, 2013.
- K. Luzuriaga et al., “Viremic relapse after HIV-1 remission in a perinatally infected child,” N Engl J Med, 372:786-88, 2015.
- SPARTAC Trial Investigators et al., “Short-course antiretroviral therapy in primary HIV infection,” N Engl J Med, 368:207-17, 2013.
- J.P. Williams et al., “HIV-1 DNA predicts disease progression and post-treatment virological control,” eLife, 3:e03821, 2014.
- J.H. Elliott et al., “Activation of HIV transcription with short-course vorinostat in HIV-infected patients on suppressive antiretroviral therapy,” PLOS Pathog, 10:e1004473, 2014.
- N.M. Archin et al., “Administration of vorinostat disrupts HIV-1 latency in patients on antiretroviral therapy,” Nature, 487:482-85, 2012.
- O.S. Søgaard et al., “The HDAC inhibitor romidepsin is safe and effectively reverses HIV-1 latency in vivo as measured by standard clinical assays,” 20th International AIDS conference, Melbourne, Australia, July 20–25, 2014.
- T.A. Rasmussen et al., “Panobinostat, a histone deacetylase inhibitor, for latent-virus reactivation in HIV-infected patients on suppressive antiretroviral therapy: a phase 1/2, single group, clinical trial,” Lancet HIV, 1:e13-e21, 2014.