TAG HIV Basic Science, Vaccines, and Cure Project Blog

Study Questions Role of HDAC Inhibitors in HIV Cure Research

The Keystone Symposia conference “Frontiers in HIV Pathogenesis, Therapy and Eradication” is taking place this week in Whistler, Canada. A poster presentation at the meeting by Jana Blazkova from the laboratory of Anthony Fauci at the National Institute for Allergy and Infectious Diseases (NIAID) (abstract #108) seems likely to place a proverbial cat among the pigeons when it comes to the use of HDAC inhibitors as a strategy to reverse latent HIV infection. HDAC inhibitors are a class of drugs that target cellular components involved in the repression of gene activity, and many studies have reported that they can cause expression of HIV proteins by latently infected, resting CD4 T cells. At the HIV Persistence Workshop in December 2011 and at CROI earlier this month, preliminary results from a small phase I clinical trial of the HDAC inhibitor vorinostat (also known as SAHA) in people with HIV on antiretroviral therapy (ART) were reported by researcher David Margolis. Analysis of cell-associated HIV RNA expression from large pools of resting CD4 T cells sampled from six participants indicated that a single 400mg dose of vorinostat led to a significant, roughly 5-fold increase compared to baseline measures, with the effect being relatively consistent (ranging from 1.5 to 10-fold). The results generated considerable press coverage due to being widely viewed as a first step toward disrupting HIV latency and perhaps curing the infection.

Jana Blazkova’s poster presentation offers data that suggests that the impact of HDAC inhibitors may be far more limited than has been hoped. Blazkova and colleagues—including latency expert Tae-Wook Chun from NIAID—sampled resting CD4 T cells from 27 individuals with suppressed viral loads on long term ART, and then tested the ability of different compounds to induce expression of HIV RNA by latently infected cells in the laboratory. Three HDAC inhibitors were included: vorinostat, valproic acid and oxamflatin. A pan-stimulator of T cells, anti-CD3 antibody, was used as a control along with prostratin, a compound which activates latent HIV via a different mechanism than HDAC inhibitors. The study found that, after exposure to the HDAC inhibitors, the amount of virion-associated HIV RNA in culture supernatants (the fluid in the culture outside of the CD4 T cells) was not significantly different from that seen when no treatments were added. In contrast, there was the expected significant increase in the amount of HIV RNA in the supernatants after addition of anti-CD3 and prostratin. 

Additional analyses showed that there was no correlation between the amount of HIV RNA detected after exposure to HDAC inhibitors and the frequency of resting CD4 T cells containing HIV DNA. For both anti-CD3 and prostratin, the correlation between these two measures was statistically significant. Comparison of the envelope gene sequences between the HIV RNA and HIV DNA also showed that the RNA detected after HDAC inhibitors appeared to be inducing a very limited subset of the diverse sequences that were detectable in the latent HIV DNA.

Tae-Wook Chun included a description of the findings in a talk at the conference on Wednesday evening but, surprisingly, the data did not prompt any questions from Margolis or any of the other scientists in attendance who are studying HDAC inhibitors as potential reversers of HIV latency. Chun took pains to cite the importance of Margolis’s work in placing the goal of purging the HIV reservoir back onto the research agenda, and stated his hope that perhaps the in vivo results will trump the observations of his laboratory.  In the absence of a discussion, the explanation for the divergent findings is unclear, although one difference is that Margolis is measuring cell-associated HIV RNA in his trial whereas Blazkova et al looked at virion-associated HIV RNA in culture supernatants; perhaps it is possible the bulk of the RNA induced by the HDAC inhibitors did not exit the infected cells. If the primary goal is to make the latently infected cells visible to the immune system, as was suggested by studies presented at CROI, it might not matter so much if the cells do not produce many virions because intracellular production of HIV proteins should be sufficient to cause presentation of HIV antigens to CD8 T cells. However, in the absence of effective immune responses against the latent HIV reservoir displaying any viral antigens, it appears unlikely that HDAC inhibitors could cause sufficient virion production to trigger HIV-induced cytopathic effects and thus kill infected resting CD4 T cells. This conclusion is supported by the findings reported by Robert Siciliano’s laboratory at CROI (covered in a prior blog post), which showed that functional CD8 T cell responses are needed to eliminate latently infected resting CD4 T cells after exposure to vorinostat. Although it may be possible to address this issue by bolstering CD8 T cell responses, if HDAC inhibitors only awaken HIV in a small subset of latently infected cells—the other main finding of this new work—that will be a much thornier problem to solve. 

Blazkova’s study is in press at the Journal of Infectious Diseases and it is likely that there will be further discussion of the data once it is published.

UPDATE: As per the comment below by Livia Pedroza Martins, the paper has now been published: 

J Infect Dis. (2012) doi: 10.1093/infdis/jis412

First published online: June 25, 2012

Effect of Histone Deacetylase Inhibitors on HIV Production in Latently Infected, Resting CD4+ T Cells from Infected Individuals Receiving Effective Antiretroviral Therapy

Jana Blazkova1,3, Tae-Wook Chun1,3,4, Bietel W. Belay1, Danielle Murray1, J. Shawn Justement1, Emily K. Funk1, Amy Nelson1, Claire W. Hallahan1, Susan Moir1, Paul A. Wender2 and Anthony S. Fauci1

1National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892, USA

2Department of Chemistry, Stanford University, 337 Campus Drive, Stanford, CA, 94305, USA

3 Contributed equally.

Abstract

Persistence of the latent viral reservoir has been recognized as a major obstacle to eradicating HIV in infected individuals receiving antiretroviral therapy. It has been suggested that histone deacetylase inhibitors (HDACis) may purge HIV in the latent viral reservoir. However, the effect of HDACis on the degree and extent of HIV expression in the latent viral reservoir has not been fully delineated. Here, we demonstrate that HDACis do not induce HIV production in the latent viral reservoir of aviremic individuals. Therefore, alternative therapeutic strategies may be necessary to eliminate HIV in the latent viral reservoir.

Inching Forward with HIV Vaccines: Digesting Envelope Decoys

At the recent CROI in Seattle, antibody expert Dennis Burton compared HIV’s envelope structure to an assemblage of Tootsie Pops. In this analogy, the parts of HIV’s envelope that are susceptible to neutralizing antibodies are represented by the chocolate contained inside the Tootsie Pop, and the carbohydrate molecules that act as decoys (by inducing ineffective non-neutralizing antibody responses) are represented by the coating of candy that surrounds the chocolate filling. Last year on the blog I wrote about two studies exploring a new strategy for dealing with HIV’s shielded envelope: using certain enzymes to digest—essentially eat away—the carbohydrate covering so that neutralizing antibody targets are revealed. One of the research groups pursuing this idea is that of James Binley at Torrey Pines Institute for Molecular Studies, and in a new paper in the Journal of Virology they report that they have now refined the approach sufficiently to create unshielded envelope proteins (known as trimers) that can be tested in animal models. The hope is that these envelope proteins will be able to reliably induce neutralizing antibodies, unlike the carbohydrate-clad versions that have been tested to date. 

First published February 2012, doi: 10.1128/​JVI.06938-11

HIV-1 Virus-Like Particles Bearing Pure Env Trimers Expose Neutralizing Epitopes but Occlude Non-Neutralizing Epitopes

Tommy Tong1, Ema T. Crooks1, Keiko Osawa1 and James M. Binley1 

1Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, CA 92121, USA

ABSTRACT

Hypothetically, since native HIV-1 Env trimers are exclusively recognized by neutralizing antibodies, they might induce them in a vaccine setting. This idea has not been evaluated, due to the difficulty of separating trimers from non-functional Env (uncleaved gp160 and gp41 stumps). The latter are immunodominant and induce non-neutralizing antibodies. We previously showed that non-functional Env can be selectively cleared from virus-like particle (VLP) surfaces by enzyme digests (Crooks ET, Tong T, Osawa K, Binley JM. J.Virol. 85: 5825 (2011)). Here, we investigated the effects of these digests on the antigenicity of VLPs and their sensitivity to neutralization. Before digestion, WT-VLPs (bearing wild type Env) and UNC-VLPs (bearing uncleaved gp160) were recognized by various Env-specific mAbs, irrespective of their neutralizing activity, consistent with the presence of non-functional Env. After digestion, only neutralizing mAbs recognized WT-VLPs, consistent with selective removal of non-functional Env (i.e. “trimer VLPs”). Digests eliminated the binding of all mAbs to UNC-VLPs, again consistent with removal of non-functional Env. An exception was mAb 2F5 that weakly bound to digested UNC-VLPs and bald VLPs (bearing no Env), perhaps due to lipid cross-reactivity. Trimer VLPs were infectious and their neutralization sensitivity was largely comparable to undigested WT-VLPs. However, they were ∼100-fold more sensitive to mAbs 4E10 and Z13e1, suggesting increased exposure of the gp41 base. Importantly, a scatterplot analysis revealed a strong correlation between mAb binding and neutralization of trimer VLPs. This suggests that trimer VLPs bear essentially pure native trimer that should allow its unfettered evaluation in a vaccine setting.

19th Conference on Retroviruses & Opportunistic Infections

The 19^th annual Conference on Retroviruses & Opportunistic Infections (CROI) took place last week from Monday through Thursday in Seattle. As has consistently been the case in recent years, the organizers have done a commendable job in making the conference accessible online: all sessions—including poster discussions—are available via webcast, and very few presenters have failed to make their slides available. 

Research into curing HIV infection featured prominently at the meeting, building on the case of Timothy Brown that was reported for the first time on a poster at CROI four years ago. A symposium on the subject was held from 4-6pm on Wednesday March 7^th and featured several excellent overview presentations on the state of the science. The major message that emerged from data presentations on cure-related research was that a one-two punch may be needed to address HIV reservoirs: firstly, dormant HIV in latently infected cells must be awakened and, secondly, effective T cell responses are likely to be required to deliver the coup de grace and kill the HIV-infected cells that are induced to produce virus.  

The rationale for this two-pronged approach was encapsulated in presentations by David Margolis from the University of North Carolina and Liang Shan from Johns Hopkins University. Margolis showed that administration the HDAC inhibitor vorinostat (also known as SAHA) successfully induced latently infected cells to express HIV RNA in six trial participants on ART (up from the four participants Margolis was able to discuss last December at the HIV persistence workshop). Liang Shan described laboratory experiments demonstrating that HIV-specific CD8 T cells from individuals with chronic HIV infection are generally unable to kill CD4 T cells that are induced to produce virus by exposure to vorinostat. In many cases, this failure could be overcome by stimulating the HIV-specific CD8 T cells with HIV antigens immediately prior to mixing them with the HIV-infected CD4 T cells, suggesting to Shan and colleagues that therapeutic immunization should be combined with anti-latency drugs (the paper containing these results was published by the journal Immunity on the same day as Shan’s CROI talk, see abstract below).

Although the goal of finding and killing latently infected cells is central to cure research efforts, it is not applicable to all strategies under study. Gene modification approaches that aim to make susceptible cells resistant to HIV—and thereby potentially give the immune system the upper hand against the virus—continue to be studied, with Sangamo BioSciences in the lead with their approach that abrogates expression of the HIV co-receptor CCR5 on CD4 T cells. Currently, the delivery of Sangamo’s treatment requires removal of CD4 T cells by apheresis, followed by modification and expansion of the cells in the laboratory and reinfusion into the individual. Another technique that is being explored in non-human primates involves gene modification of stem cells, which give rise to CD4 T cells and other cell types and therefore might be a better target. The research group of Hans-Peter Kiem at Fred Hutchinson Cancer Research Center in Seattle presented a “proof of concept” study showing that a gene encoding an HIV entry inhibitor (C46) could be introduced into stem cells in macaques, ultimately leading to substantial levels of circulating gene-modified CD4 T cells (~20%). When these animals were challenged with an SIV/HIV hybrid SHIV virus, CD4 T cell levels dipped but recovered and the proportion of gene gene-modified CD4 T cells increased. Interestingly, unmodified CD4 T cells also appeared to be protected from infection, leading the researchers to speculate that gene-modified CD4 T cells were contributing to more effective virus-specific immune responses in the treated macaques. Kiem’s group plans to conduct similar studies using Sangamo’s technology to disrupt the CCR5 gene in stem cells.

Online sources of CROI 2012 coverage include: 

AIDSMap

AIDSMeds

HIVandHepatitis.com

IFARA

MedPageToday

NATAP

The Body

 

Immunity. 2012 Mar 7. [Epub ahead of print]

Stimulation of HIV-1-Specific Cytolytic T Lymphocytes Facilitates Elimination of Latent Viral Reservoir after Virus Reactivation.

Shan L, Deng K, Shroff NS, Durand CM, Rabi SA, Yang HC, Zhang H, Margolick JB, Blankson JN, Siliciano RF.

Department of Medicine, Johns Hopkins University School of Medicine, Baltimore,

MD 21205, USA; Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA. 

Abstract

Highly active antiretroviral therapy (HAART) suppresses HIV-1 replication but cannot eliminate the virus because HIV-1 establishes latent infection. Interruption of HAART leads to a rapid rebound of viremia, so life-long treatment is required. Efforts to purge the latent reservoir have focused on reactivating latent proviruses without inducing global T cell activation. However, the killing of the infected cells after virus reactivation, which is essential for elimination of the reservoir, has not been assessed. Here we show that after reversal of latency in an in vitro model, infected resting CD4(+) T cells survived despite viral cytopathic effects, even in the presence of autologous cytolytic T lymphocytes (CTLs) from most patients on HAART. Antigen-specific stimulation of patient CTLs led to efficient killing of infected cells. These results demonstrate that stimulating HIV-1-specific CTLs prior to reactivating latent HIV-1 may be essential for successful eradication efforts and should be considered in future clinical trials.