HIV Cure Strategiesa
Why Should We Try to Cure HIV?
Although current antiretroviral therapy (ART) is highly effective at controlling HIV-1 replication, it does not eradicate or cure the infection. There are several compelling reasons for trying to cure HIV-1. First, despite efforts to expand access to treatment, the majority of HIV-1-infected individuals worldwide do not receive ART, which leads to ongoing transmission of the virus. Second, because current ART does not eradicate HIV-1, infected patients must take ART for many decades, which may eventuate in difficulties with adherence, substantial cost, and the potential for long-term side effects. Third, HIV-1-infected patients have increased rates of cardiovascular disease, liver disease, neurocognitive disorders, and other non-infectious complications, which may be driven by elevated levels of inflammation that persist despite suppressive ART. Finally, HIV-1 infection continues to be associated with stigma and social isolation, which adversely affect quality of life. Given the limitations of current ART, there is a renewed and concerted effort to find a cure for HIV-1.
Although complete viral eradication, or a “sterilizing cure,” is the ultimate goal, the concept of a “functional cure” has been introduced, which includes strategies aimed at achieving host control of the virus without the need for ART. Several clinical observations within the past few years have fueled the belief that one or the other of these types of “cure” might be possible. Certainly, the most compelling example is that of the “Berlin patient,” the only person to have been cured of HIV-1. This HIV-1-positive patient with virologic suppression on ART received, as treatment for acute myelogenous leukemia, allogeneic hematopoietic stem cell transplants from a donor who carried a homozygous deletion in CCR5 (Hutter, 2009), the co-receptor for HIV-1, thereby making his new CD4+ T cells resistant to infection. Following discontinuation of ART, no HIV-1 RNA has been detected in the Berlin patient’s peripheral blood; moreover, multiple attempts to detect HIV-1 RNA or proviral DNA in cellular reservoirs and other tissue compartments have been negative (Yukl, 2013). Because of the risk of stem cell transplantation, however, this intensive approach is not appropriate in HIV-1-infected patients who do not have a hematologic malignancy. Another notable “proof-of-concept” derived from studies of early initiation of ART during acute HIV-1 infection. The Visconti study identified 14 HIV-1-infected patients whose viremia has remained controlled for years after the interruption of ART that had been initiated during primary infection (Saez-Cirion, 2013). Along these lines, the “Mississippi baby,” a child born to an HIV-1-infected mother, was started on ART 30 hours after delivery (Persaud, 2013) and quickly achieved virologic suppression. The child was lost to follow-up, however, and ART was discontinued by the caregiver. Despite stopping ART, the virus remained undetectable for 27 months, but the child ultimately experienced virologic rebound (Luzuriaga, 2015). These examples demonstrate that it is possible, under extraordinary circumstances, to eradicate HIV-1 (in the case of the Berlin patient) or control HIV-1 without ART (in the case of the VISCONTI cohort and, temporarily, the Mississippi child). Now, the challenge is to extend the insights from these remarkable cases to the development of practical interventions that will lead to ART-free remission in the large population of people living with HIV-1.
Early Establishment and Persistence of the Latent HIV-1 Reservoir
In 1995, Chun et al. identified integrated provirus as a persistent reservoir of infection in the resting CD4+ T cells of HIV-1-infected patients (Chun, 1995). Although most activated memory CD4+ T cells are destroyed during viral replication, a small fraction of infected cells survive to return to a resting and memory state. Once converted to a resting memory state, HIV-1 gene expression is shut down, resulting in latently infected CD4+ T cells (Nabel, 1987). Because these infected cells do not express viral proteins, they remain hidden from the host immune response; moreover, without active replication, antiretroviral drugs cannot act against the virus. Although infected resting cells leave the quiescent memory pool at a steady rate, the pool of infected cells persists, perhaps in part because of homeostatic proliferation. It has also been suggested that specific CD4+ T cell memory subsets, including central memory (TCM), transitional memory (TTM), and memory stem cells (TSCM), harbor the majority of integrated HIV-1 DNA and that eradication therapies may require targeting of specific CD4+ T cell populations (Buzon, 2014).
When latently infected cells are reactivated, viral gene expression is renewed and productive infection is reignited. In patients on long-term ART, the frequency of latently infected cells is extremely low: Less than 1 per 1 million resting memory CD4+ T cells harbors replication-competent HIV-1 (Finzi, 1997; Wong, 1997). Nevertheless, this latent pool decays very slowly: The mean half-life of this reservoir is approximately 44 months, and as a result, suppressive ART would need to be maintained for more than 60 years to achieve viral eradication even if an infected person has only 100,000 latently infected cells (Finzi, 1999). In addition, it is conceivable that latent infection may persist in cells that are not CD4+ T cells; however, this has yet to be proven.
It was initially believed that early suppression of viral replication during primary infection might prevent the reservoir from becoming established. However, Chun et al. demonstrated that ART initiated within 10 days of primary infection did not prevent the generation of latently infected CD4+ T cells (Chun, 1998), pointing toward an early seeding of the reservoir. Newer data from the rhesus macaque model demonstrate that the latent reservoir is established within days of virus exposure, even before virus can be detected in peripheral blood (Whitney, 2014); the implication of this finding is that it will be practically impossible to treat or even diagnose HIV-1 infection early enough to avoid reservoir seeding. Several studies, however, have demonstrated that initiating ART during the acute/early phase of the infection results in a smaller HIV-1 reservoir (Ananworanich, 2012; Hocqueloux, 2013; Saez-Cirion, 2013), suggesting that early treatment could be beneficial by reducing the barrier to cure (Henrich, 2013; Strain, 2005).
The presence and persistence of the HIV-1 latent reservoir represent the major obstacle for cure approaches. For this reason, a deeper understanding of how latency is maintained and how this state can be reversed is critical to inform HIV-1 eradication strategies (Richman, 2009).
“Kick and Kill”
In order to achieve viral eradication, or at least a state of HIV-1 suppression without requiring continuous ART, different strategies have been proposed, including modification of the host immune response to achieve enhanced control of viral replication, interventions to prevent reactivation of virus latency (Mousseau, 2015), and gene therapy to increase the resistance of target cells to HIV-1 infection (Tebas, 2014). Currently, the strategy that is receiving the most attention is the “shock and kill” or “kick and kill” approach. In this strategy, the first step is to flush out HIV-1 from the latent reservoir by activating proviral DNA expression in resting cells, leading to de novo viral protein production (the so-called “shock” or “kick”). If this “kick” is successful, the next step is to enhance immune recognition and elimination of infected cells (the “kill”). This two-step approach, however, requires a latency-reversing strategy and an antiviral immune response in order to clear infected cells; both tasks are encumbered by substantial challenges.
Latency Reversal Approaches
Immune Enhancing Strategies
Although latency reversal will be crucial for eradication strategies, inducing viral replication alone will most likely not be sufficient to eliminate the infection. Indeed, in an in vitro model, reversal of latency alone did not result in clearance of infected cells (Shan, 2012). For this reason, it is anticipated that following reactivation, cells harboring the reservoir will need to be actively cleared, most likely through a second line of attack by the host’s immune system. Strategies to enhance immune responses via immunization or immunomodulation have been proposed based on the hypothesis that boosting T cell responses will lead to enhanced viral control—similar to that in so-called HIV-1 elite controllers, patients who maintain undetectable viral loads in the absence of ART (Deeks, 2007), in whom antiviral T cells have been associated with viral suppression (Deeks, 2007; McMichael, 2010).
The ability to enhance the host’s immune responses by therapeutic vaccination faces several key challenges. The majority of HIV-1-infected individuals have dysfunctional HIV-1-specific effector cells as a result of continuous antigenic stimulation prior to treatment (Sauce, 2013), and ART only incompletely restores T cell functionality. Furthermore, in patients who initiate ART during chronic infection, almost all of the proviral sequences in the latent reservoir contain escape mutations that prevent killing of infected cells by cytotoxic T lymphocytes (Deng, 2015; Papuchon, 2013). The implication is that an effective vaccination strategy, instead of just expanding preexisting responses that already had failed to control the infection, would need to improve the quality and functionality of HIV-1-specific immune response and elicit CD8+ T cell responses against previously untargeted epitopes or unmutated regions of the virus to avoid escape. To achieve this goal, multiple approaches are currently being tested in preclinical and clinical studies, including the following:
• Viral vector-based vaccines, such as adenovirus, poxvirus modified vaccinia Ankara, and modified cytomegalovirus: Some of these approaches to deliver HIV-1 antigens have demonstrated robust immunogenicity, inducing broad and durable cellular immune responses that were able to protect monkeys against SIV infection in preclinical challenge studies (Barouch, 2012, 2013; Hansen, 2011, 2013).
• Dendritic cell-based vaccines to deliver HIV-1 antigens: In one study, this approach was associated with reduced plasma viral load post-treatment interruption (Levy, 2014).
Some of these vaccines have been tested already in humans and are safe and immunogenic, but proof of efficacy in reducing the HIV-1 reservoir has yet to be demonstrated.
The recent identification of novel broadly neutralizing anti-HIV-1 antibodies (bNAbs), which are able to neutralize the majority of viral strains at very low concentrations, may provide another approach to target the HIV-1 reservoir. In preclinical studies, administration of bNAbs was shown to reduce plasma viremia in chimeric simian–human immunodeficiency virus (SHIV)-infected macaques (Barouch, 2013; Shingai, 2013); in fact, one monoclonal antibody, PGT121, also resulted in substantial reductions of proviral DNA in peripheral blood, lymph nodes, and gastrointestinal mucosa (Barouch, 2013). Two bNAbs have been tested so far in HIV-1-infected humans and have shown promising reductions in plasma viremia (Caskey, 2015; Lynch, 2015). However, it remains to be determined what effect bNAbs will have on the viral reservoir in humans, and limitations such as the lack of accessibility of antibodies to certain anatomic reservoir sites (e.g., the central nervous system) will need to be overcome.
Finally, a novel method to combine antibody and T cell activity against HIV-1-infected cells is through bispecific protein constructs, which are designed to latch onto HIV-1 envelope proteins on the surface of infected cells while also binding to CD3 on T cells. This approach directs cytotoxic T cells to eliminate infected cells while obviating the need for the T cells to specifically bind to HIV-1 surface antigens (Pegu, 2015; Sung, 2015). Early in vitro studies of this approach are promising, but additional preclinical work is needed to confirm that these immunomodulatory proteins are safe enough to test in human trials.
Rendering the Host’s CD4+ T Cells Resistant Against Infection
The finding that the Berlin patient appeared to be cured of HIV-1 after receiving a stem cell transplant from a CCR5-δ32 homozygous donor has inspired attempts to generate HIV-1-resistant cells through gene therapy. Using artificial restriction enzymes such as zinc finger nucleases (ZFN), DNA can be cleaved at specific sites; this approach has been used to disrupt the CCR5 gene (which encodes the HIV-1 co-receptor) in CD4+ T cells (Perez, 2008). Using the ZFN strategy, Tebas et al. modified the CCR5 gene ex vivo in autologous CD4+ T cells in 12 HIV-1-infected subjects and infused the cells back into the autologous donor (Tebas, 2014). The study found that genetically modified cells persisted in vivo with a half-life of nearly 1 year. Although no dramatic difference was seen in viral load set points following interruption of ART in 6 study participants, the modified cells appeared to be protected from HIV-1 infection because unmodified cells showed a faster depletion.
Excising the HIV-1 Provirus from the Host Cell Genome
Several research groups have successfully applied this technology to excise HIV-1 provirus from the host cell genome (Ebina, 2013; Hu, 2014; Liao, 2015). Importantly, the disruption of provirus expression not only restricted transcriptionally active provirus but also blocked the expression of latently integrated provirus (Ebina, 2013). Moreover, inserting the stably expressed CRISPR/Cas9 system into a T cell line conferred long-term protection against HIV-1 infection (Liao, 2015). These results from in vitro cell culture models are promising, and this technology may open new avenues to developing antiviral therapies in the future.
Although antiretroviral medications are able to effectively treat HIV-1 infection, there are many compelling reasons to attempt to cure HIV-1, including the stigma and isolation experienced by many infected patients. The major barrier to HIV-1 cure is the persistence of a long-lived population of latently infected cells in patients on suppressive treatment; because the latent reservoir is established soon after HIV-1 acquisition, even early initiation of ART cannot cure the infection. Current efforts to cure HIV-1 infection are centered on flushing HIV-1 out of the latent reservoir along with enhancing immune mechanisms to clear infected cells. We are still in the early days of this massively difficult undertaking, and it is too soon to determine whether the approaches being pursued now will be effective. However, just as the development of combination ART was based on a series of advances that culminated in our ability to successfully treat HIV-1, the stepwise progress being made today, it is hoped, will lead us to an even greater breakthrough: the capability to eradicate or control HIV-1 without the need for lifelong therapy.
Questions and Answers
This chapter in Fundamentals of HIV Medicine has accompanying questions that can be answered for continuing medical education (CME) credit. To access these questions, visit www.cmeuniversity.com and enter course ID 11635 in the “Find Post-test/Evaluation by Course” field. Access to CME credit expires April 2018.
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a This chapter is based on a previous version written by David Margolis MD, University of North Carolina at Chapel Hill.