Workshop Report and Commentary

Table of Contents

• Introduction
• Workshop Report and Commentary
• Day Two
• Day Three: Margolis Reaches a Milestone, the Crowd Thins for Functional Cures
• References

Introduction

This meeting had a limited numbers of attendees and brought together an impressive group of leading researchers.

The abstract book and late breaker abstracts are available in PDF format from the conference website and links:

www.hiv-workshop.com/workshop-2011.htm

www.hiv-reservoir.net/index.php/the-news/189-abstract-book-2011-hiv-persistence-workshop.html

The site also contains daily rapid summaries of the workshop that will be followed in the next few weeks by more detailed reports.

Workshop Report and Commentary

Inaugurated in 2003, the bi-annual International Workshop on HIV Persistence during Therapy (aka "the persistence workshop") is the brainchild of researcher Alain Lafeuillade. The meeting presaged the recent explosion of interest in pursuing a cure for HIV infection, a pursuit many had considered quixotic until the case of Timothy Brown came to light in 2008.

As has been extensively documented, Brown's apparent cure resulted from a debilitating odyssey of treatments required for the grim diagnosis of acute myelogenous leukemia, enhanced with a mix of insight and good fortune on the part of his doctor Gero Hutter, who was able to provide a stem cell transplant from a donor lacking the major HIV co-receptor CCR5.

The sea change wrought by this fortuitous "proof of concept" was much in evidence at the 2011 persistence workshop this past December; the tentative forays into basic science that were once emblematic of the field are now mixed together with more ambitious plans for advancing ideas into the clinic. Perhaps most strikingly, two large pharmaceutical companies -- Gilead and Janssen/Tibotec -- described their use of industrial scale screening to search for compounds that are active against latent HIV; this represents an unprecedented expansion of efforts once confined to under-resourced academic labs.

A number of online resources are available with information on presentations at the 2011 persistence workshop: Lafeuillade runs a website called the Reference Portal on HIV Reservoirs & Eradication Strategies which includes an expanding number of reports, video interviews and commentary.¹

David Margolis from the University of North Carolina has written a comprehensive report for Jules Levin's National AIDS Treatment Advocacy Project (NATAP) website.² Jon Cohen also covered one the most notable presentations in the journal Science.³

This report and commentary represents my subjective take on events.

To try and briefly summarise the top-line stories that emerged from the 2011 meeting:

• A triumvirate of researchers -- Courtney Fletcher, Mario Stevenson and Tim Schacker -- presented data suggesting that sporadic, very limited rounds of HIV replication may occur in some individuals on ART due to poor penetration of certain drugs into the lymphoid tissues. However, preliminary data were only available from a small number of participants (~4-5) so the implications are still uncertain. According to the clinicaltrials.gov entry for the study, it is now expanding from the original enrollment target of 12 to 40 so additional information should soon be forthcoming.⁴ Alain Lafeuillade has posted an interview with Mario Stevenson about the findings, and these presentations were the subject of Jon Cohen's story in Science.⁵
• An Italian research group led by Andrea Savarino described a retrospective analysis involving 18 rhesus macaques infected with SIVmac251 that participated in various studies combining ART with drugs targeting the viral reservoir. The analysis found an association between the number of "anti-reservoir" drugs animals received and the likelihood of controlling SIV to undetectable levels after ART was interrupted; however only three macaques controlled SIV to this degree so the findings should be considered very preliminary. The workshop organisers issued a press release about the data suggesting that for the first time they show that anti-reservoir drugs may be able to contribute to what is now frequently referred to as a "functional cure" (control of viral load in the absence of ART). In an interview with Alain Lafeuillade, Savarino is careful to note that the findings require confirmation in human studies because they could relate to unknown factors specific to the three macaques that controlled SIV in the experiment.⁶ This caveat is underscored by the fact that there are relatively few studies involving ART treatment of SIVmac251 in macaques to provide context, and in those that have been published there appear to be some examples of animals that spontaneously controlled viral load after ART interruption (both in control groups and in recipients of a DNA-based therapeutic SIV vaccine).
• David Margolis from the University of North Carolina presented the first data on the use of a histone deacetylase (HDAC) inhibitor named SAHA (aka vorinostat) in individuals with HIV. HDAC inhibitors are at the forefront of efforts to pharmaceutically urge HIV out of latency, so news from Margolis's trial has been eagerly awaited. While very preliminary, and derived from just four participants, the results so far suggest that the approach is able to increase HIV expression by latently infected cells. It took Margolis many years to get the trial started due to concerns about the safety of HDAC inhibitors (which are used as cancer treatments and can cause serious toxicities) but no serious side effects have occurred to date. As Margolis stressed, much more work is needed before any conclusions can be drawn about the promise of the approach.
• The burgeoning involvement of the pharmaceutical industry in cure-related research -- represented by presentations from Romas G from Gilead and Roger Sutmuller from Janssen/Tibotec -- was important news because it promises to transform the drug discovery effort by increasing the number of compounds that are being screened by many orders of magnitude.

The workshop agenda was divided into discrete topic areas spread over three days. The first session addressed the subject of animal models, and was led off by Jeff Lifson from the National Cancer Institute (NCI) at Frederick who has nearly two decades of experience studying SIV infection in rhesus macaques. Lifson outlined some of the considerations in developing an appropriate model for cure-related studies, which include mimicking the degree of viral suppression achieved with ART in humans and developing tools to comprehensively assess the impact of additional interventions on SIV reservoirs.

The models currently in use include:

• Macaques infected with hybrid SIV/HIV viruses encoding HIV reverse transcriptase (SHIV-RT), treated with efavirenz, emtricitabine and tenofovir
• Macaques infected with SIVmac251 or SIVmac239 treated with multi-drug regimens (e.g., tenofovir, emtricitabine, raltegravir and ritonavir-boosted darunavir +/- maraviroc)
• Pigtailed macaques infected with SIV/17E-Fr and SIV/Delta B670 treated with tenofovir, integrase inhibitor, saquinavir, atazanavir (this model is primarily being used to assess issues relating to viral activity in the brain)

Lifson described a study conducted by his laboratory in which macaques were infected with the highly virulent challenge virus SIVmac239 and, after sixteen weeks, treated with a multi-drug antiretroviral regimen comprising an integrase inhibitor, tenofovir, emtricitabine, and ritonavir-boosted darunavir. Suppression of viral load to less than 30 copies/mL was eventually achieved, but Lifson noted that it took longer than is seen with HIV in humans. Like the vast majority of macaque studies, the experiment involved Indian rhesus macaques, and Lifson suggested that viral load suppression might be easier to achieve in Chinese rhesus macaques (this subspecies has been shown to control SIV somewhat better in the absence of ART). Lifson acknowledged that refinement of the SIV/macaque model for cure-related research is ongoing, and he cautioned against the premature adoption of any one approach as a standard. As an example of the pitfalls of premature standardisation, he cited the HIV vaccine field's mistake in adopting a SHIV89.6p challenge model that turned out to have essentially no relevance to human HIV infection.

One potentially important new technology that Lifson highlighted is called digital PCR, which is vastly superior to traditional PCR for measuring small quantities of nucleic acid in samples. PCR amplifies nucleic acid sequences from a single sample by inducing rounds of copying of the original sequence, then back-calculating how many were originally present using a formula that takes into account the number of rounds of copying; however these calculations can be imprecise for a number of reasons. Digital PCR divides a sample into many discrete "microfluidic" wells and then uses PCR to look for the nucleic acid sequence of interest in each well, providing a readout as to whether the sequence is absent (0) or present (1). The total amount of nucleic acid sequence that was present is then calculated based on the number of negative and positive wells, using an approach called a Poisson distribution. Digital PCR assays have only recently been commercialised and a number of laboratories are now busy using them to measure SIV and HIV in research studies.

The presentations following Lifson illustrated the diversity of animal models in use, and the uncertainties associated with them. Andrea Savarino from the Istituto Superiore di Sanità in Rome provided an update on experiments conducted by his group involving macaques infected with SIVmac251. In a paper published in AIDS last year, Savarino and colleagues reported that the gold-based rheumatoid arthritis drug auranofin reduced the reservoir of SIV-infected cells in animals treated with combination ART.⁷

At the workshop, Savarino presented results of a retrospective analysis of 18 macaques (including those included in the experiments reported in the paper) that have received various combinations of antiretrovirals and "anti-reservoir" drugs including auranofin and buthionine sulfoximine (BSO). The breakdown of the antiretroviral regimens employed was as follows:

• ART: tenofovir, emtricabine, raltegravir
• Intensified ART (iART): tenofovir, emtricabine, raltegravir, ritonavir-boosted darunavir
• Mega-ART: tenofovir, emtricabine, raltegravir, ritonavir-boosted darunavir, maraviroc

Three of the 18 macaques have controlled SIVmac251 to undetectable (<40 copies/mL) levels after interruption of all treatment for several months, and Savarino reported that there was a significant correlation between the number of "anti-reservoir" drugs received and this salutary outcome (for the purposes of this analysis, the CCR5 inhibitor maraviroc was counted as an anti-reservoir drug due to evidence that it reduced the amount of SIV DNA when added to intensified ART and preliminary results from a human study suggesting it may impact reservoirs). Some macaques also received the HDAC inhibitor SAHA, but an impact on the SIV reservoir could not be demonstrated.

The complicated sequence of treatments and outcomes in the three macaques that have controlled viral load off ART can be roughly summarised as follows:

• Macaque P252: ART, ART+auranofin, iART+auranofin, iART+SAHA, iART+auranofin, treatment interruption, viral load control to limit of detection, viral load rebound, Mega-ART, treatment interruption, viral load control, viral load rebound, viral load control, viral load rebound, Mega-ART, viral load control, viral load rebound, Mega-ART+BSO, viral load control (100+ days)
• Macaque P157: ART, iART, Mega-ART+auranofin+BSO, treatment interruption, viral load rebound, viral load control (~60 days), viral load blip, viral load control (~50+ days)
• Macaque P177: ART, iART, Mega-ART, Mega-ART+auranofin, treatment interruption, viral load rebound, Mega-ART, treatment interruption, viral load rebound, viral load control, viral load rebound, viral load control (~50+ days)

The data appear encouraging but there are some potential caveats:

• The model of SIVmac251 infection treated with combination ART (the drugs used in the study included tenofovir, emtricabine, raltegravir, ritonavir-boosted darunavir and maraviroc) is not well characterised, at least in terms of the published literature
• There were very few control animals, and the results are not from a single study but rather from multiple experiments, sometimes involving the same macaques being rolled over from prior experiments
• As can be seen from the sequence of events in the three controlling macaques, the treatments were complex and there was variability between animals in terms of exactly when different interventions were administered

As Savarino stresses in his video interview with Alain Lafeuillade, human trials are now required to ascertain if the macaque results can be translated to HIV.

Paul Luciw presented results of an experiment in which macaques infected with SHIV-RT had prostratin and valproic acid added to long-term ART (efavirenz, emtricitabine and tenofovir) prior to an interruption. Luciw showed evidence of reduced viral RNA and DNA in tissues but when treatment was interrupted there was no significant difference in viral load rebound compared to macaques treated with ART alone. Daria Hazuda from Merck has included several of Luciw's slides in her recent presentations on cure research so the main findings can be viewed online, however note that prostratin is only referenced as a "protein kinase C activator" and valproic acid as an "HDAC inhibitor."⁸

Luciw also mentioned that he repeated the experiment adding raltegravir to the ART regimen and in that case there was no additional viral RNA and DNA reduction in tissues resulting from the anti-reservoir drugs, but he was running out of time and was unable to give any details; this finding is perhaps a reminder of how much uncertainty still surrounds macaque models for cure research.

Jerome Zack is trying to make drug-delivery nanoparticles out of weird cellular particles called "vaults" made of three proteins and a bit of RNA.⁹ Zack presented some preliminary evidence that they can be engineered to deliver potential latency activators prostratin and bryostatin, Zack is also working with Paul Wender at Stanford to develop better analogues of these drugs to use. The goal is to come up with some lead vault-delivered anti-latency compounds to test in the BLT humanised mouse model.

Shifting topics to the virological aspects of HIV persistence, Sarah Palmer from the Karolinska Institute reported results of an intensive evaluation of viral genetics pre-ART and on long-term ART (up to >12 yearrs) in 12 people (seven treated at acute infection, five during chronic infection) to look for evidence of viral evolution that would be indicative of ongoing replication. No evidence suggestive of HIV replication was found in various CD4 subsets and other cell types in blood, lymph tissue, bone marrow and gut. Palmer noted that no hematopoetic progenitor cells (HPCs) containing HIV DNA could be found; occasional positive signals from HPC samples turned out to be due to low-level contamination with CD4 cells. This finding was recently echoed in a paper from Bob Siliciano's group at Johns Hopkins.¹⁰

Palmer drew attention to one case where a large amount of HIV DNA containing a huge deletion encompassing all of the protease gene was discovered. Since HIV can't replicate without protease, this demonstrates that the division of CD4 T cells carrying integrated, non-functional proviral HIV DNA can contribute to what may appear to be an HIV reservoir by some measures (but really isn't because the virus is defective). Mario Stevenson coined the term "junkyard DNA" for these non-functional proviruses, and it was quickly adopted at the workshop.

Tae-Wook Chun from the National Institute of Allergy and Infectious Diseases (NIAID) offered some data suggesting HDAC inhibitors may not be all they're cracked up to be in terms of reversing HIV latency, in the hands of his lab they didn't induce a significant amount of viral RNA from latently infected cells compared to prostratin (which is a potent activator generally considered too toxic for human use). Chun also said that the latently infected cells induced to produce viral RNA don't seem to die ("we haven't seen any evidence of cell death'), suggesting that induction using HDACs might have little effect in the absence of an immune response capable of killing the infected cell.