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:

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

http://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

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:

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

http://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.

Day two

Day two of the persistence workshop featured the presentations from industry, with Romas Geleziunas from Gilead and Roger Sutmuller from Janssen/Tibotec talking back-to-back about the ongoing work at their companies.

Gilead is looking at both virus activators and immune modulators, with Romas Geleziunas seemingly already having taken on board what Tae-Wook Chun had suggested the previous day: reactivating latent infection may not be enough to kill a cell, hence immune mechanisms may need to be induced to deliver the coup de grace. Geleziunas described Gilead’s high throughput primary cell screening assay, which is a modified version of an assay developed by Vincente Planelles and Alberto Bosque. [11]

So far they’ve identified three HDAC inhibitors from the Gilead drug library, imaginatively named 001, 002 and 003. 001 is 10-fold more potent than SAHA but inhibits all classes of HDACs (which I think is a bit of a worry from a toxicity perspective) while 002 is of interest because while less potent it doesn’t score positive on the AMES test (the standard test for assessing mutagenic potential). 001 and 003 were both AMES positive. Rats tolerated 3 weeks of 002 in a preliminary safety study. Romas noted that HDAC inhibitors only activate a fraction of the virus expression seen with pan-activating CD4 T cell stimulation using CD3 and CD28 antibodies, raising the question of whether the HDAC inhibitors are only activating a proportion of the latently infected CD4 cells, or rather causing less virus expression per cell. This question remains to be resolved.

High throughput screening of a Gilead library and a commercially available drug library produced a 1% hit rate, identifying 89 compounds that could be grouped into 15 clusters based on their structures. One was a calcium pump inhibitor named thapsigargin, possibly after a character from Lord of the Rings. It was a ‘robust activator’ of latency in cells from 6 out of 6 donors. Romas didn’t say anything more about it and Wikipedia offers an explanation as to why: “It is a tumor promoter in mammalian cells”. Another was a “broad spectrum nonspecific tyrosine kinase inhibitor” called tyrphostin A which worked on cells from 3/6 donors. Since they hadn’t expected to find kinase inhibitors, they then tried screening a library of those and got a 20% hit rate. Evidence of activity at low concentrations and dose responses were seen. Next steps are to confirm activity with more selective kinase inhibitors and explore the signaling pathways that are causing these compounds to work.

Switching to the topic of bolstering immunity, Romas said Gilead is looking at a TLR7 agonist it has in development for hepatitis B. It’s been tested in chimps and woodchucks, where it has shown antiviral activity and dose-dependent induction of alpha interferon production and T cell and B cell activation. In woodchucks, it led to induction of antibodies against the hepatitis B surface protein. A small phase I human study has been safely conducted, also showing evidence of some T cell and B cell activation. Next step is to study the impact on HIV-infected cells and potentially test it in animal models in combination with HDAC inhibitors.

Meanwhile the overarching goals of Gilead’s program continue to be:

• More high throughput screening
• Uncover novel mechanisms (e.g. as may happen as a result of the identification of kinase inhibitors)
• Discover new chemical entities (NCEs).

Roger Sutmuller from Janssen/Tibotec then described his company’s efforts which have not been discussed publically before. He outlined the basic goal of discovering safe and effective compounds to reactivate latent HIV i.e. those that cause little or no cell activation and ideally have the potential to be combined. Unlike Gilead, Tibotec starts with a Jurkat cell line assay to identify compounds, after which they have a preplanned set of steps involving evaluation of:

• Toxicity/immune stimulation
• Virus reactivation in primary T cell assays
• Virus reactivation in latently infected cells from HIV+ individuals ex vivo
• Medicinal chemistry selection of lead compounds
• Testing in a humanised mouse model developed by Roberto Speck
• Testing of the pathways involved in drug activity eg using microarrays, HIV mutants with various signaling elements disabled, short-interfering RNAs etc.

Using the Jurkat cell line assay, 35,000 compounds have been screened to date, and the next step is to screen 480,000 compounds from a Johnson & Johnson ‘diversity library.’ Of those screened to date, 800 HDAC inhibitors have popped out (a 20% hit rate), 25 protein kinase C agonists (a family prostratin belongs to) and 600 unknowns that can be grouped into 11 different ‘chemotypes.’

Sutmuller went on to describe their in-house primary T cell assay, which involves fresh cells expanded in the lab and infected with an HIV encoding green fluorescent protein (GFP). Cells are rested to create latency and then drug activity is measured based on the extent to which the cells light up green. They’re using this assay to screen medium sized libraries; it can handle about 2,000 compounds per week. He showed some data from one compound ‘229,’ which induced virus at about half the level of pan-stimulator PMA, and worked even better in combination with SAHA. The next step is to study these and other compounds in Roberto Speck’s humanised mouse model, which involves 3TC and TDF given in food pellets and a long-acting version of TMC 278 that is delivered by weekly injection. They have seen good viral suppression and can recover latently infected CD4 cells using this system.

Among the other highlights from day two, Una O’Doherty from the University of Pennsylvania showed that CD8 T cells from elite controllers can kill what appear to be latently infected CD4 cells because they express the HIV Gag protein, just with much slower kinetics than seen with activated CD4 cells (and without causing spreading infection). O’Doherty suggested that perhaps this means latently infected CD4 cells aren’t as invisible to the immune system as has been thought, which provoked some controversy because—as she happily acknowledged—it is not yet known whether the same holds true for latently infected CD4 cells from individuals on ART.

In an effort to hone in on which elements of the Berlin patient’s treatment were necessary to achieving the apparent cure of HIV infection, the ever-curmudgeonly John Mellors (University of Pittsburgh) presented an analysis of ten people who had undergone myeloablative chemotherapy and autologous stem cell transplants for lymphoma. None of these individuals were cured of HIV infection, leading Mellors to conclude that in the case of Timothy Brown, the CCR5-negative transplant was important, possibly along with the graft-versus-host disease Brown experienced. In the Q&A afterwards, workshop attendee Mike McCune from UCSF suggested that total body irradiation (TBI) might also have played a role.

Santiago Moreno (Hospital Ramon Y Cajal, Madrid, Spain) presented some preliminary evidence that the CCR5 inhibitor maraviroc may activate a protein complex named NF-kappaB when the drug binds to the CCR5 receptor. Because NF-kappaB activation can stimulate latent HIV, Moreno suggested that maraviroc might have anti-reservoir activity, as was previously suggested by a small uncontrolled pilot study conducted by Moreno’s laboratory and reported at a symposium prior to the 2010 International AIDS Conference in Vienna. However, results from a randomised trial of ART intensification with maraviroc were debuted at the persistence workshop by Maria Puertas, and this study was unable to document any additional declines in HIV reservoirs associated with receipt of the drug (HIV DNA levels fell by ~8-fold in both arms).

In a session on acute HIV infection, Marty Markowitz from Aaron Diamond AIDS Research Center presented 96-week results from a 3-drug vs. 5-drug treatment study, showing essentially no significant differences in a variety of reservoir and immunological measures in blood and gut. There was a slight reduction in cell-associated HIV RNA levels at week 96 in the 5-drug group but Markowitz felt this was unlikely to be meaningful. Jintanat Ananworanich (HIV Netherlands Australia Thailand Research Collaboration) described a study involving treatment of people with very, very early HIV infection, in which 60 people have so far been enrolled, with an average time from screening to enrollment of just 3 days. This would not seem like much time for someone to process the news that they have become HIV infected and make a decision to enter a trial involving a multiple treatments and sampling from the peripheral blood, CNS and GI tract, but Ananworanich said “acceptance rates are quite high.” Individuals were in what in the following Fiebig stages of seroconversion:

• 34% stage I: within 5 days of infection
• 9% stage II: within 10 days of infection
• 48% stage III: within 13 days of infection
• 9% stage IV: within 19 days of infection

24-week results on a subset of participants indicated significantly smaller reservoirs in blood and gut of stage I vs. III or IV, with total and integrated HIV DNA being undetectable in a proportion of the earliest-treated individuals.

The very last presentations of day two involved the tag team of Timothy Schacker (University of Minnesota), Courtney Fletcher (University of Nebraska) and Mario Stevenson (University of Miami) outlining very preliminary results from their small study of viral replication in anatomical and cellular reservoirs. A total of 12 individuals are enrolled, ART naive at baseline but then treated (mostly with TDF, FTC and ritonavir boosted atazanavir) and analysed regularly up to six months. Not all individuals have data available yet, and the number of individuals from whom data were reported varied between the different presenters. Courtney Fletcher looked at drug levels in nine people, finding that some drugs (particularly atazanavir, FTC and efavirenz) may not reach adequate levels in lymph nodes and gut. Mario Stevenson then showed that in some study participants, 2-LTR circles increased in lymph tissue after starting ART, in one case along with a rise in proviral DNA. In one other individual, levels of both 2-LTR circles and proviral DNA went down. Stevenson stated: “this does not necessarily denote ongoing replication” but proposed an alternative model in which a population of long-lived cells can generate virions that infect one more cell and that’s it – just one cycle of replication, in other words. He stated this would not lead to viral evolution but could replenish the latent reservoir. In the Q&A, John Coffin from the NCI got up to the microphone and noted that since latency is a rare event in infected cells, and since Stevenson was saying these were single-cycle rounds of infection, the number of times latency would be created is not known, and may well not be often enough replenish the reservoir.

Timothy Shacker closed out the talks with a description of his efforts to correlate Fletcher’s and Stevenson’s results with measurements of viral RNA on the follicular dendritic cell (FDC) network in lymph tissue (using in situ hybridisation). Schacker created 3D graphs for several participants that included 2-LTR circle levels, DNA levels, levels of viral RNA on FDCs and, lastly, drug levels. There appeared to be correlations between the various measures, but how many people had evidence of ongoing HIV replication cycles was unclear. Schacker noted that there was a significant inverse correlation between levels of FTC diphosphate in lymph tissue and viral RNA on FDCs. Additional results from the expanded version of this study are needed in order to understand if this is a broadly applicable phenomenon, and whether poor tissue penetration of antiretrovirals represents an under-appreciated obstacle to curing HIV infection.

Day three: Margolis reaches a milestone, the crowd thins for functional cures

The major news on day three of the workshop was the presentation by David Margolis (University of North Carolina) of very preliminary results from the phase I/II study of the HDAC inhibitor vorinostat (SAHA). The trial has a complicated schema, largely due to the safety concerns of the FDA regarding the drug, which scores positive on the AMES mutagenic test (a red flag for regulators even though the significance is not fully understood).

The first step of the protocol involved screening potential participants to assess whether vorinostat could reactivate latent HIV from their CD4 T cells ex vivo. Thirteen individuals had ~4 billion lymphocytes extracted by leukopheresis, then sorted into discrete pools of 1 million purified resting CD4 cells each (ending up with 24-36 pools per participant). These pools were exposed to either vorinostat or no drug, and a mean level of HIV RNA per million cells (and a standard deviation) was calculated for each person (the assay used can measure down to 10 copies per million cells). Margolis noted that the statistical approach used to calculate the mean RNA levels is robust but complicated, and a paper explaining it is currently in press at an unnamed statistics journal.

Four of the thirteen people screened showed a statistically significant upregulation of HIV RNA expression in this analysis and were therefore recruited into the next step of the trial. A 200mg dose of vorinostat was given first for safety, followed by a 400mg dose to study pharmacokinetics and for analyses of histone acetylation and acetylation of the p21 gene (in other words, analyses of the effects of the drug on cellular genetic machinery and not HIV). The pharmacokinetic data mirrored that reported in cancer studies and cellular acetylation (both total and p21 gene) was maximal by 8 hours then trended down by 24 hours.

A final 400 mg dose of vorinostat was then administered with leukopheresis performed 4-6 hours afterward based on the pharmacokinetic data indicating this would be around the time of maximum activity. No grade 1 or greater toxicities were seen, and HIV RNA expression increased compared to baseline in all four individuals by a mean of 4.4-fold (range: 3-6.6 fold). HIV RNA in peripheral blood was also assessed using a single copy assay but no change was detected, perhaps not surprisingly given that this was a single dose study.

Margolis was obviously very encouraged by the data and stated that they had successfully “demonstrated induction of full length HIV RNA expression within a window of time after a single vorinostat exposure.” He concluded that obstacles to HIV RNA expression can overcome “at least in some cells.” But he stressed that many questions remain, including:

• Is there an equal effect to multiple doses or does it become attenuated?
• How much exposure is needed?
• Should drug be administered continuously or pulsed?
• Will toxicities emerge?
• What number of cells is needed to measure relatively rare reactivation events?
• Does RNA expression lead to virion production or clearance of cell?
• Are additional inducers needed?
• Are additional interventions needed to clear the latently cells that have been induced to express HIV RNA?

The final session of the meeting was on functional cures. Dishearteningly, the crowd of attendees thinned noticeably but the first presenter, Paula Cannon, was undeterred. “This is the first time people are going to be talking about functional cures,” she opened sunnily. “I know you’re all very obsessed with the reservoirs but we don’t really care about the reservoir – if there’s a little bit of virus left in the body, so what?” Having stuck fear into the hearts of any remaining reservoir obsessives, she then outlined what she meant, highlighting three key goals for those in pursuit of a functional cure:

• Reducing the pool of HIV target cells and thereby reducing the harmful immune activation and inflammation that is central to pathogenesis.
• Creating HIV-resistant HIV-specific CD4 T cells.
• Taking advantage of HIV as a selection agent to drive the expansion of resistant cells.

Cannon went on to review the Sangamo zinc finger nuclease (ZFN) approach to deleting CCR5, the work conducted by her laboratory to adapt it to modify hematopoietic stem cells (HSCs), and the efficacy demonstrated in a published experiment in which humanised mice were engrafted with the CCR5-deleted stem cells and challenged with HIV. Work is now underway to advance the approach into HIV positive people who need stem cell transplants as treatment for lymphoma, in collaboration with John Zaia and David DeGusto from City of Hope who have previous experience of studying gene-modified HSCs in this setting. Cannon explained that preparation for the trial has involved switching from relatively easy-to-use HSCs obtained from fetal cord blood to rather more uncooperative adult stem cells. These cells are called mobilised peripheral blood stem precursor cells (mPSCs) and sampling involves giving G-CSF for four days then conducting apheresis to extract white blood cells, followed by ex vivo purification of CD34+ cells. This procedure has now been performed on 13 donors, obtaining 42 billion white blood cells of which around 0.5% were CD34+ cells; Cannon estimates that around 1% of the CD34+ cells are ‘true’ stem cells. These mPSCs are now being used in mouse studies to address a number of issues prior to human testing.

One such experiment assessed whether pre-existing immunity to adenovirus might be problematic, because an adenovirus vector is used to deliver the zinc finger nuclease into the mPSCs. Mice were given a high titer of anti-adenovirus antibodies prior to delivery of the mPSCs and, encouragingly, no difference was seen in the extent of engraftment compared to controls given phosphate buffered saline (PBS).

Next steps include large scale tumorigenicity studies in ‘NOD scid gamma’ (NSG) mice and evaluation of modified mPSC under ‘maximising’ conditions to test the upper limit of on and off target effects (there is some evidence that ZFNs can disrupt genes other than the CCR5 target, particularly a similar region of the CCR2 gene). Mice given the maximised mPSCs will be kept for many months and extensively analysed for safety.

Following Paula Cannon, Carl June gave an update on the use of the same technology to modify CD4 T cells that are extracted from individuals with HIV using apheresis, expanded and modified in the laboratory, and reinfused into the same individual. Previous presentations of data from these phase I trials has generated considerable excitement, because the proportion of modified CD4 T cells persisting in the blood and gut of participants far exceeds the extremely modest levels obtained with prior gene therapies delivered using the same approach. Significant CD4 T cell count increases have also been documented out to nine months of follow up. Unusually, CD4:CD8 ratios have also significantly improved from an average of 0.5 at baseline to 1.5 at last analysis; this type of improvement is rarely observed as a result of ART, and may have implications in terms of improving long-term health because inverted CD4:CD8 ratios are a well-documented risk factor for illness in the HIV-uninfected elderly.

Most intriguing, however, is a trial involving a 12-week analytical treatment interruption (ATI). Data is now available from six individuals who have undergone the ATI and while all experienced a viral load rebound, levels began falling prior to the reinitiation of ART, which June noted was not the case in a prior gene therapy study involving an ATI (an evaluation of a candidate named VRX496).

One notable individual controlled viral load to below the level of detection (<50 copies/mL) before ART was restarted. This person turned out to be heterozygous for the delta32 CCR5 deletion, which means that the ZFNs could work more efficiently because only one CCR5 gene in each cell had to be disrupted in order for CCR5 expression to be completely abrogated (instead of two as is normally the case). Importantly, June found a significant correlation between the proportion of modified CD4 T cells and viral load control during the ATI. This suggests that an antiretroviral effect is achievable with the approach, and that the potency of the effect may be boosted if the proportion of modified cells can be increased.

In the Q&A period, June was asked if he had assessed whether gene-modified HIV-specific CD4 T cells may have contributed the viral load results; he replied that HIV-specific CD4 T cell responses have not yet been analyzed in the ATI trial.

The last two talks in the final workshop session addressed the development of methods that attempt to specifically target latent HIV and excise it from the DNA of infected cells (or damage the provirus in order to render it non-functional). On paper, at least, these approaches sound very appealing but it was clear that significant hurdles remain. Jan van Lunzen (University Medical Centre Hamburg-Eppendorf) discussed the modification of an enzyme called Cre recombinase to target HIV DNA. The modified version, dubbed Tre recombinase, has successfully excised proviral DNA from cells in vitro and work is now underway to study how it might be delivered. Next steps involve studies in humanised mice using a lentiviral vector to deliver the Tre recombinase to CD34+ stem cells; the vector is designed to be ’self-inactivating’ in cells that do not contain HIV DNA. As an aside, Jan van Lunzen also mentioned a patient of his who started ART during early infection, was treated for five years, then stopped six years ago, had a small viral load blip and has been undetectable ever since. HIV RNA cannot be found in blood, gut or CNS. According to van Lunzen, the individual has a “very strong HIV-specific CD4 response,” and he highlighted the case as being similar to Christine Rouzioux’s report of five individuals treated very early who have controlled viral load to undetectable levels off ART for an average of around five years. [12] These case reports may bode well for prospects for a functional cure, van Lunzen suggested.

Keith Jerome from the Fred Hutchinson Cancer Research Center recounted the efforts of his group to employ different enzymes, endonucleases, to target latent HIV. The idea in this case is to induce mutations in the HIV provirus in order to render it non-functional. Some success has been achieved in vitro but considerable challenges remain in terms of improving the efficiency of targeting and developing delivery methods that might be able to get the endonucleases to where they are needed. Jerome’s work is now being supported by a Martin Delaney Collaboratory grant from NIH.

The last word at the 2011 persistence workshop was given to Nobel laureate Françoise Barré-Sinoussi, who outlined the International AIDS Society’s development of a Global Scientific Strategy ‘Towards an HIV Cure’ and encouraged audience members to attend an IAS symposium on the subject that will take place in Washington DC immediately ahead of the 2012 International AIDS Conference. Barré-Sinoussi also stressed the importance of the work and the need to continue the momentum which has placed curing HIV infection back at the top of the research agenda.

The 6th International Workshop on HIV Persistence, Reservoirs & Eradication Strategies is scheduled for 2013 in Miami.

References

1. Reference Portal on HIV Reservoirs and Eradication Strategies website.
   http://www.hiv-reservoir.net/
2. Margolis D. HIV Persistence during Therapy 5th International Workshop. natap.org, 2011.
   http://www.natap.org/2011/HIV/122111_01.htm
3. Cohen J. Tissue says blood Is misleading, confusing HIV cure efforts. Science 23 December 2011: Vol. 334 no. 6063 p. 1614.
   http://www.sciencemag.org/content/334/6063/1614.summary
4. Clinical Trial. Tissue drug levels of HIV medications.
   http://clinicaltrials.gov/ct2/show/NCT01490346
5. Mario Stevenson Interview. (9 December 2011).
   http://www.youtube.com/watch?v=_fhb95p__9g
6. Andrea Savarino Interview on HIV Cure. (10 December 2011).
   http://www.youtube.com/watch?v=xIqdDU_OEFU
7. Lewis MG et al. Gold drug auranofin restricts the viral reservoir in the monkey AIDS model and induces containment of viral load following ART suspension. AIDS. 25(11):1347-1356, July 17, 2011.
   http://journals.lww.com/aidsonline/toc/2011/07170
8. Hazuda D. Powerpoint can be downloaded at the bottom of this page, Luciw’s data is on slides 21-25):
   http://www.iasociety.org/Default.aspx?pageid=416#session5
9. TEDxUCLA – Leonard Rome – online video. (29 August 2011).
   http://tedxtalks.ted.com/video/TEDxUCLA-Leonard-Rome-Vaults-mo
10. HIV-1 DNA is detected in bone marrow populations containing CD4+ T cells but is not found in purified CD34+ hematopoietic progenitor cells in most patients on antiretroviral therapy. J Infect Dis. (2012). First published online: 24 January 2012.
    http://jid.oxfordjournals.org/content/early/2012/01/24/infdis.jir884.abstract
11. Bosque A et al. Studies of HIV-1 latency in an ex vivo model that uses primary central memory T cells. 2011 Jan;53(1):54-61. Epub 2010 Oct 21.
    http://www.ncbi.nlm.nih.gov/pubmed/20970502
12. Hocqueloux C et al. Long-term immunovirologic control following antiretroviral therapy interruption in patients treated at the time of primary HIV-1 infection. 19 June 2010 – Volume 24 – Issue 10 – p 1598-1601.
    http://journals.lww.com/aidsonline/Fulltext/2010/06190/Long_term_immunovirologic_control_following.27.aspx

There is currently an interest in using boosted protease inhibitor monotherapy as a maintenance strategy in resource rich countries.

A pilot substudy of the DART trial, Second-line Anti-Retroviral therapy in Africa (SARA), randomised 192 patients who had received 24 weeks of lopinavir/ritonavir-based (LPV/r) second-line combination therapy to either continue on this combination or to receive LPV/r maintenance monotherapy. Prior to the switch, DART patients had taken first line therapy for a median of 4.4 years.

Data were presented as a late breaker poster at IAS 2010 suggesting few differences between the two groups in CD4 increases or adverse events in the short term. [1] At week 24 the mean CD4 gain was 48 vs 42 cells/mm3 in the combination and monotherapy arms respectively. For those completing 72 weeks the gains were 159 vs 153 cells/mm3.

No real-time virology was performed in DART but plasma samples were stored from: time at switch to second line, time of SARA randomisation, 24-weeks after SARA randomisation and latest time point (35-107 weeks after SARA randomisation).

Dave Yirrel presented results at Glasgow 2010 from a retrospective analysis of viral load, measured by Roche Amplicor v1.5 on the stored samples. In addition, genotype resistance was assessed on samples with viral load >1000 copies/mL at 24 weeks. Analyses were intent to treat. [2]

The median CD4 counts overall were 86 cells/mm3 at switch to second line and 245 cells/mm3 at SARA randomisation. The majority of patients (86%) had received a triple nucleoside first line regimen and the remainder two nucleosides and an NNRTI. At SARA randomisation 22% were receiving LPV/r + 2/3 NRTIs, 15% LPV/r + NNRTI and 62% LPV/r + NNRTI + NRTI. Of those with viral load measurements 135/173 (77%) had viral load <50 copies/mL.

The investigators found a higher proportion with undetectable viral load among patients on combination therapy compared to monotherapy at week 24, p=0.007. They reported 77% (70/91) vs 60% (66/94) had viral loads <50 copies/mL and 94% vs 84% had viral load <1000 copies/mL.

Among the small number of patients for whom 96-week data were available for analysis, the proportion with rebound to >200 copies/mL was greater in the monotherapy than combination therapy arm: 50% vs 20% (n=7 per arm). This difference was similar among those with rebound >1000 copies/mL: 40% vs 10% (n=7 in the monotherapy and , n=8 in the combination therapy arms).

Genotype results from the patients with viral load >1000 copies/mL at 24 weeks showed 0/5 patients with major protease inhibitor mutations of those in the combination therapy arm and 4/16 (of 19 patients with rebound to 1000 copies/mL) in the monotherapy arm.

The investigators concluded that, over the relatively short period of follow up (median 60 weeks) since SARA randomisation, there was an increase in low level viraemia with monotherapy compared to combination therapy, but no evidence of systematic increase in viral load after loss of suppression.

The EARNEST trial due to start next year will provide data on the long-term effectiveness of PI maintanance monotherapy in this setting. [3]

comment

Neither the numbers involved, nor the duration of the trial make it possible to make any definite conclusion from these data. But it does seem that boosted PI monotherapy may not be a viable option in settings without access to viral load monitoring.

References:

1. Gilks CF et al. Boosted protease inhibitor monotherapy as maintenance Second-line Anti-Retroviral therapy in Africa: a randomised controlled trial (SARA). 18th International AIDS Conference. 18-23 July, 2010. Vienna. Poster abstract LBPE16.
   http://pag.aids2010.org/Abstracts.aspx?AID=17479
2. Pillay et al. Virological findings from the SARA trial: boosted PI monotherapy as maintenance second-line ART in Africa. 10th International Congress on Drug Therapy in HIV Infection, November 7-11. Glasgow. Oral abstract O20. Published in Journal of the International AIDS Society 2010 13(Suppl 4). O214.
   http://www.jiasociety.org/content/13/S4/O20
3. Europe – Africa Research Network for Evaluation of Second-line Therapy (EARNEST) study.
   http://earnest.cineca.org/index.php

A poster discussion session included studies looking at different aspects of dual infection and reinfection and included two case studies that showed where this had a clinical outcome. Two other studies, one from London and one from San Francisco, reported on aspects of dual infections.

Erika Castro from University of Vaudois Hospital, Lausanne, and colleagues presented a useful case of reinfection between two men (M1 and M2) who had been sexual partners since 2006. [1]

M1 was initially diagnosed in 2000 during primary infection and had been suppressed on HAART through to 2007 with no history of drug resistance. M2 had been on HAART for five years with detectable viral load (range 3–4 log) and documented triple class resistance. In February 2008, viral load in M1 rebounded to 280 copies/mL and continued increasing. Resistance and phylogenetic tests were compared from 2000 and 2008 (86 sequences:  whole-genome (n=28), env (n=28), and gag (n=25).

All sequences were sub-type B. The genotypic analysis from 2008 showed 25 new related drug resistance mutations in M1 (11 in RT and 14 in protease), of which 23 were also present in M2. Additionally, M1-2008 sequences clustered within the M2-2008 branches and distinct from M1-2000 sequence clusters in all trees. Recombination between the original M1 and M2 strains was not observed, with M1 being replaced with M2 sequences following superinfection.

The case is important for highlighting several aspects of HIV reinfection:

• That reinfection can occur in established infection.
• That it can occur between regular partners (that no immune protection develops to repeated exposure to one virus).
• That reinfection can occur after several years of exposure (as with initial infection, chance and probability are low).
• That there is a clinical risk from reinfection when partners have different resistance profiles (most reinfection cases are only discovered because of unexplained viraemia in stable patients).
• That ART did not offer protection against reinfection, probably because any PEP effect would be negligible if the new MDR virus was resistant to the ARVs in that combination.

Martine Braibant from University de Tours Hospital and colleagues presented another case of sub-type B being reinfected with subsequent sub-type B infection. [2]

This patient entered a long-term non-progressor (LTNP) cohort in 1995 aged 58, following a ten year history of HIV infection, with a CD4 count >600 cells/mm3 for the previous 5 years and viral load of 135 copies/mL on study entry. From 1995–1999, viral load slowly increased to around 10,000 copies/mL and CD4 count dropped steadily to <500 cells/mm3. The initial infection was found to have a 20 nucleotide deletion in nef (consistent in 28 sequences) and the loss of viral control and immunologic progression from 1995 was associated with detection of subsequent sequential reinfection with two fully competent phylogentically different strains. Both new strains were also sub-type B.

The patient responded well to HAART, achieving viral suppression and CD4 recovery >700 cells/mm3 within the first year of therapy, but potentially would have maintained the option to remain off-treatment for many years if reinfection hadn’t occurred.

This case highlighted that:

• Progression rates may be determined by both virologic and immunologic factors
• Reinfection in the absence of resistance may have clinical implications on disease progression, requiring earlier treatment
• That long-term exposure to low level sub-type B virus did not promote an immune response that was protective of subsequent sub-type B infections,

The authors highlighted in their conclusion that this last point showed the inherent difficulties for development of a preventative vaccine.

Jane Deayton and colleagues from St Barts Hospital, London report three cases of inter-clade dual infection detected during routine genotype testing. The three cases were Caucasian UK-born MSM whose only risk factor was sexual exposure in the UK. Case 1 had been diagnosed in 2001 and received a genotype test in 2008 prior to starting therapy that indicated dual infection with sub-types B and G. Cases 2 and 3 were tested after their HIV diagnoses in 2007 and 2008 and showed dual infections with B and CRF02_AG, and B and A, respectively. Both these patients were reported to be stable off treatment.

None of the cases included significant drug resistance mutations.

The authors concluded that these were the reports of cross-clade dual infection from sexual transmission in MSM in the UK are rare (only one other was known). Additionally this indicated onward transmission of clades associated with African epidemics and an increasing cross-over of viruses between different demographic groups.

Finally, Larry Bragg and colleagues in San Francisco presented a poster looking at cases where majority viruses change in an individual though competitive expression following dual infection. Whilst difficult to distinguish from superinfection (ie reinfection after an initial infection) sequential expression of dual infections (SEDI) theoretically could come from dual initial infection or reinfection shortly after initial infection, especially prior to seroconversion. [4]

The group included 220 recently infected persons with at least two genotypes (560 person-years of follow-up). The mean age was 37 and median time from infection to first test was 107 days. Divergent viruses appeared in 7 cases, an overall incidence density of 1.24/100 person-years.

Their model estimated a risk of SEDI that was 16-fold higher in the first year post-infection compared to after one year. The estimated rate of SEDI was 4.1/100 person-years (95% CI, 1.8 to 9.2) in the first year following infection and was 0.2 per 100 person-years beyond 1 year post-infection (95% CI, 0.03 to 1.8).

This study highlighted that in the case of dual infection or early reinfection the predominant infection is determined within the first year.

References

1. Castro E et al. HIV-1 superinfection with a drug-resistant strain in a patient successfully controlled with ART. Poster abstract 480.

http://www.retroconference.org/2010/Abstracts/37374.htm

2. Braibant M et al. Disease progression after intrasubtype superinfection in an HLA-B57+ asymptomatic LTNP initially infected with a nef-defective HIV-1 strain. Poster abstract 302.

http://www.retroconference.org/2010/Abstracts/38615.htm

3. Deayton J et al. Inter-clade dual HIV-1 infection: an emerging phenomenon. Poster abstract 447.

http://www.retroconference.org/2010/Abstracts/38460.htm

4 Bragg L et al. HIV-1 Superinfection Surveillance in an Acute Infection Cohort Using pol Sequences from Resistance Genotyping: 1996 to 2008. Poster abstract 446.

http://www.retroconference.org/2010/Abstracts/39292.htm

The only criticism I had of the Paris meeting, apart from the perennial rudeness of French waiters, was that some of the parallel sessions became so full that there was only standing room (sometimes five deep) and since my advanced age no longer allows me to stand for more than 15 minutes without calf pain I retreated into rooms that normally I would not visit. Thus I found myself in a fascinating series of presentations on viral dynamics, chaired by Mark Wainberg and Jean-Luc Darlix.

Firstly, a group from the Institut Pasteur and The University of Saarland in Germany showed data on quantifying HIV DNA from the reverse transcription to integration so essentially trying to work out how many virions are needed to infect a new cell [1]. They showed in an elegant series of experiments that the intracellular enzyme proteasome degrades up to 75% of incoming virions. Combining this data with other studies the conclusion was that the fraction of HIV RNA that gets converted into provirus could be as low as 1%. Thus, since the spleen cells they examined had on average 3-4 proviruses each, it suggests that 300-400 virus particles are required for the infection of an individual cell. They also showed that there is rampant recombination of viruses with up to 29% difference between viruses within an individual cell. Doug Richmann questioned if this data could be validated in cells within infected individuals whilst Vincent Calvez from Paris suggested that cell to cell spread of virus may be more important explaining why the switch from non-syncytial inducing (R5) virus to syncytial inducing (X4) virus may herald a rapid decline in health.

This led nicely onto the second presentation from the team of Eric Arts at Case Western Reserve University, which, working with the Institute of Tropical Medicine in Antwerp, is following a cohort of patients over a protracted period of time to evaluate the relationship between HIV fitness and viral genetic diversity [2].

It is known that several factors have an impact on progression of disease; viral load, possession of nef, use of either CCR5 or CXCR4 co-receptor. In this study they used growth competition assays to compare the fitness of viruses from 12 individuals followed for 2-5 years as well as assessing the genetic diversity of the HIV quasispecies by sequencing a section of the envelope of at least 10 clones from the same patient sample used to estimate the fitness. What they showed was that as time passed the viruses within an individual become gradually fitter and their genetic diversity (variability) and divergence from the original strain increase. So for example: Patient K was followed over six years during which his T4 count dropped from 800 to 400 with a concomitant rise in viral load of 1 log. Four isolates were examined over this time period, which showed a doubling of viral fitness with a rise in diversity and divergence. Across the 12 patients there was a marked correlation between ex vivo fitness and genetic diversity (p<0.003). When HAART was commenced, there was a reduction in viral fitness that paralleled the drop in viral load and rise in T4 count.

HIV viral fitness was also correlated with time from seroconversion (r=0.08; p<0.001). The presenter questioned whether treatment might reduce the fitness of the virus. Doug Richmann suggested that resistance might be a simpler explanation. However, none was found in the samples examined. This study raises the question of why the epidemic is not accelerating if fitness is rising constantly from time of infection. Much as in the previous abstract, this may be a reflection of a genetic bottleneck in the cellular level during transmission, which reduces fitness back to lower levels. A further question was asked about what co-receptor these viruses were using. The answer was all R5 except for the last sample of Patient K where the virus had become dual tropic, fitting well with the idea that time is an important factor in determining co-receptor usage and disease progression. What this study suggests is that perhaps it may be better to treat early to reduce viral fitness, diversity and the speed of progression, a nice idea if we had completely non-toxic therapy.

A study from Albany Medical College and the New York State Department of Health examined the dynamics of R5 and X4 viruses after the commencement of HAART in eight individuals (six female) [3]. In patients with X4 virus there was a marked suppression of these viruses within two weeks, from the blood, whilst the trajectory of clearance of R5 virus was much slower. A shift in co-receptor usage in the female genital tract also occurred but the dynamics differed from the blood due to R5 viruses persisting for a much longer period. These changes did not seem to be linked to the density of particular co-receptors, cellular activation or cytokine production. Work on characterising any resistance in these isolates is ongoing.

Jacques Reynes from Montpellier then showed data that argued, however, that the density of CCR5 receptors may be an important determining factor in HIV replication [4]. Using flow cytometric techniques, he determined the density of the CCR5 co-receptor on peripheral blood T4 cells in 23 subjects (eight women and 15 men) who underwent a 30 day interruption in HAART. T4 varied between 300 and 1739 (median 724) and their viral load was <200. CCR5 density appears to vary for around 4-24,000 molecules/cell and remains reasonably stable over time. In this study there was a strong correlation between the CCR5 expression and viral load rebound. For R5 densities <8,000 molecules/cell viral load rose to <100,000 copies/ml whereas those who rebounded to >100,000 copies/ml had densities over 8,000 molecules/cell (r=0.71; p<0.001). These results emphasise the role of CCR5 density in in vivo HIV replication. Thus, therapies such as the new CCR5 receptor blockers SCH D (Schering) and UK-427,857 (Pfizer) may offer a therapy, which not only is effective but also has a greater efficacy in those patients with the greatest potential for progression. Only clinical studies will inform as to whether this is true, however it is tantalising to hope that the arrival of these blockers may have an added advantage to those with high viral loads.

References

Unless stated otherwise, references are to the programme and abstracts of the 2nd IAS Conference on HIV Pathogenesis and Treatment, 13-16 July 2003, Paris.

1. Vartanian JP, Suspène R, Guétard D, et al. Quantitating HIV DNA from reverse transcription to integration in vivo. Abstract 146.
2. Troyer RM, Collins KR, Abraha A et al. Comparison of ex vivo HIV-1 fitness to genetic diversity during disease progression. Abstract 147.
3. Philpott S, Burger H, Kitchen C et al. Dynamics of suppression of CXCR4 specific HIV-1 strains by antiviral therapy. Abstract 149.
4. Portales P, Baillat V, Merle de Boever C. CD4+ T-cell surface CCR5 density is a predictive factor of HIV rebound after antiretroviral treatment interruption. Abstract 151.

There is little data regarding the true incidence of primary pulmonary hypertension (PPH) among HIV-positive patients. Autopsy series prior to the introduction of HAART suggested that PPH was significantly more common among HIV-positive people (incidence of 0.5%) compared to the “general population” (incidence 0.01-01%). Prognosis is extremely poor among persons with PPH and it is estimated that the survival rate among HIV-positive patients with PPH at two years is between 32-46%.

The pathogenesis of PPH is not completely understood. There is limited evidence to suggest that endothelin-1 over expression may be the primary cause of PPH. Endothelin-1 is a potent vasoconstrictor (narrows the pulmonary blood vessels). Bosentan (trade name Tracleer) is an orally administered dual endothelin-receptor antagonist that has been shown to improve exercise capacity and cardiopulmonary haemodynamics in HIV-negative patients with pulmonary arterial hypertension. This is the first study I am aware of studying its effects in HIV-associated PPH.

This is an open-label, non-comparative, multi-centre trial evaluating the efficacy and safety of bosentan in HIV-positive patients with PPH. Patients with HIV-associated PPH and NYHA functional class III received bosentan for 16 weeks (62.5 mg bid for four weeks; thereafter 125 mg bid) with either >3 months antiretroviral therapy (ART) or not on ART and CD4 count >100 cells/mm3. Patients with portal hypertension, cirrhosis or liver enzymes >3 x upper limit of normal were excluded.

Safety was assessed by CD4 cell count, viral load, liver function and adverse events. Efficacy was assessed by exercise capacity, NYHA class and haemodynamics (right heart catheterisation). The study was to enrol 30 patients; however an interim analysis of the first 10 subjects revealed significant results and enrolment was closed at 17 subjects. Results were reported on 16 subjects who completed 16 weeks of study.

Baseline characteristics of the 16 subjects are as follows: nine male, age 39 + 8 years, one HBV co-infected, three HCV co-infected, median CD4 count 333 cells/mm3, and seven subjects with HIV VL <400 copies/ml.

After eight weeks, there were no significant changes in CD4 count or the number of patients with suppression of HIV-1 RNA, suggesting no significant effect of bosentan on control of HIV infection. Adverse events included cramps (n=2), headaches (n=4), ALT/AST >3 x ULN (n=2), leg edema and/or weight gain (n=6), which improved with diuretics.

At 16 weeks, subjects improved their six-minute walk distance from 333±20 to 424±14 m (P<0.001), NYHA class (14 improved to Class I or II; two remained in Class III) and cardiac index (2. ± 0.2 to 3.4 ± 0.2l/min/m2; P<0.001). Significant decreases in pulmonary vascular resistance (781±64 to 476±64 dyn.sec/cm5; P<0.001), and mean pulmonary arterial pressure (51.7±3.4 to 43.3±3.8 mm Hg;P=0.051) were also observed. No patient died, required epoprostenol therapy or hospitalisation for pulmonary arterial hypertension during the study. One patient subsequently died although the cause of death was unknown but did involve illicit drug use. These preliminary results suggest that bosentan significantly improves PPH symptoms, functional status, exercise capacity and haemodynamics similar to those reported in the HIV seronegative population.

Bosentan appears safe when given concomitantly with antiretroviral therapy and is well-tolerated. One has to be cautious about this as the actual ART regimens were not discussed and there may be significant drug interactions as discussed below.

Nevertheless, this is extremely welcome news. I have had several patients die in the past from PPH. I have one woman who is now on bosentan for over one year and is clinically doing well. Her exercise capacity and functional status have improved remarkably. So, it is nice to see a study that supports its use. I have had concerns of potential drug interactions with bosentan. The package insert states it is a substrate of CYP2C8/9, 3A4 and induces CYP2C8/9, 3A4.

There is a large list of potential interactions as follows:

Cyclosporine: Bosentan may enhance the metabolism of cyclosporine, decreasing its serum concentrations by 50%; effect on sirolimus and/or tacrolimus has not been specifically evaluated, but may be similar. Cyclosporine increases serum concentrations of bosentan (approximately 3-4 times baseline). Concurrent use of cyclosporine is contraindicated.

Glyburide: An increased risk of serum transaminase elevations was observed during concurrent therapy with bosentan. Concurrent use is contraindicated.

HMG-CoA reductase inhibitors: Agents metabolised via CYP3A4 may be decreased by bosentan; includes atorvastatin, lovastatin, and simvastatin.

Ketoconazole: May increase the serum concentrations of bosentan; concentrations are increased approximately two-fold; monitor for increased effects. Many interactions have not been specifically evaluated, but may be extrapolated from similar interactions with inducers/inhibitors of CYP3A4 and CYP2C8/9 isoenzymes.

Key potential interactions are summarised as follows:

Anticonvulsants: Bosentan may increase the metabolism of selected anticonvulsants; includes ethosuximide, phenytoin, tiagabine, and zonisamide. The effect of concurrent therapy with enzyme-inducing anticonvulsants on bosentan concentrations has not been established.

Antipsychotics: Bosentan may enhance the metabolism (decrease the efficacy) of antipsychotics; monitor for altered response; dosage adjustment may be needed.

Calcium channel blockers: Bosentan may enhance the metabolism of calcium channel blockers, decreasing their clinical effect.

Corticosteroids: Bosentan may enhance the metabolism of corticosteroids, decreasing their clinical effect.

CYP2C9 inhibitors: May increase the serum concentrations of bosentan; includes amiodarone, fluoxetine, sulfonamides, ritonavir, zafirlukast.

CYP3A4 inhibitors: May increase the serum concentrations of bosentan; includes amiodarone, cimetidine, clarithromycin, erythromycin, delavirdine, diltiazem, dirithromycin, disulfiram, fluoxetine, fluvoxamine, grapefruit juice, indinavir, itraconazole, ketoconazole, nefazodone, nevirapine, propoxyphene, quinupristin-dalfopristin, ritonavir, saquinavir, verapamil, zafirlukast, zileuton.

Doxycycline: Bosentan may enhance the metabolism of doxycycline, decreasing its clinical effect; higher dosages may be required.

Oestrogens: Bosentan may increase the metabolism of oestrogens and reduce their efficacy.

Hormonal contraceptives: Bosentan may enhance the metabolism of hormonal contraceptives, decreasing their clinical effect; an alternative method of contraception should be considered.

Methadone: Bosentan may enhance the metabolism of methadone resulting in methadone withdrawal.

Protease inhibitors: Serum concentrations may be decreased by bosentan. Avoid concurrent use of agents metabolised by CYP3A4 or CYP2C8/9.

Warfarin: Bosentan may increase the metabolism of oral anticoagulants; monitor for changes in INR. Significant changes in INR not observed in clinical trials. In addition, the manufacturer recommends avoiding bosentan in moderate to severe hepatic impairment.

Bosentan is associated with a high incidence (11%) of significant transaminase elevations, indicating a potential for serious hepatic injury. Based on animal studies, bosentan is likely to produce major birth defects if used by pregnant women. Ideally, one would like to have pharmacokinetic studies with protease inhibitors and the NNRTIs but it is doubtful these will be done. Usually these patients are on multiple other medications, so it may be difficult to predict the various interactions and one will need to monitor these patients closely for side effects and efficacy. The real test will be whether one can show a survival benefit over time which preliminary data fortunately suggests.

Ref: Opravil M, Sitbon O, Gressin V et al. Safety and efficacy of bosentan in pulmonary arterial hypertension associated with HIV infection. Abstract 1007.

The unique co-transfection step inherent to single-cycle HIV resistance assays, can result in even relatively small amounts of wild-type virus (WT) within a viral population significantly affecting the apparent replication capacity (RC) and phenotype of mutant strains, conclude researchers from Abbott Laboratories in Illinois, USA.

The replication capacity and susceptibility of plasma isolates from patients who are off therapy or not adherent to treatment, in which wild-type virus may expand to significant levels, should be interpreted with caution, they say in an abstract presented to the Mexico resistance meeting.

In a typical single-cycle HIV phenotypic assay – the most common test for RC and phenotypic resistance – the ‘pool’ of DNA generated from patient plasma is transfected into target cells in order to capture and preserve the protease and reverse transcriptase sequence heterogeneity of the plasma virus. However, co-transfection of different viral variants into the same cell might provide the opportunity for genetic recombination or complementation, or both.

Hongmei Mo and colleagues found that four mutant constructs with different genotypes derived from dual protease inhibitor-experienced subjects receiving lopinavir/ritonavir (LPV/r) therapy displayed <5% RC when transfected alone. Co-transfection of as little as 9% of the WT clone increased the RC of the mutant clones to up to 14%. Co-transfection of a higher proportion of the WT clone further enhanced the RC of the mutants to 31–81%.

The LPV susceptibility of four mutant clones with sufficient RC for phenotypic evaluation when transfected alone ranged from 44- to 302-fold, compared to the WT clone. Incremental cotransfection of 9–50% of the WT clone decreased the LPV IC50 of the mutant clones by up to 96%, compared to WT.

Ref: Mo H, Lu L, Kempf D et al. The impact of minor populations of wild-type HIV on the replication capacity and phenotype of mutant variants in a single-cycle HIV resistance assay. XII International HIV Drug Resistance Workshop, Los Cabos, Mexico, 10-14 June 2003. Abstract 85. Published as part of Antiviral Therapy Volume 8 Issue 3.

All the approved HIV treatments so far are antiretrovirals — drugs that directly target some step in viral replication. In the future we may have another kind of treatment, immune-based therapy, which strengthens the immune system’s ability to control HIV, instead of attacking the virus directly.

A third approach, less talked about so far, might be called HIV harm-reduction treatment — preventing the virus from causing harm despite a continuing high viral load.

This could work because HIV seems to cause most of its damage indirectly — by the toxic tat protein, for example, or by dysregulation of immune responses leading them to kill normal cells — rather than by killing infected cells, which the body could normally replace. If so, then ways to block the indirect damage might become a new kind of treatment. (We distinguish HIV harm reduction from immune-based therapies because the former would not necessarily target the immune system at all — and also because it would have to be tested differently, since it might not decrease viral load, which immune-base therapies might be expected to do.)

One observation supporting this way of thinking is described in an abstract at the recent conference of the Institute of Human Virology, 9-13 September, 2002, in Baltimore. Mark Feinberg of Emory University noted that in monkeys and other primates, all known retroviral infections in their natural hosts did not cause AIDS-like disease.

But the same viruses, in primates that are not natural hosts, do cause persistent infection, loss of CD4+ T cells, and susceptibility to opportunistic infections. And in these animals that do get sick, low viral loads and strong cellular immune responses predict slower disease progression — as they do in humans with AIDS.

But at least some animals naturally infected with SIV (simian immunodeficiency virus) successfully control the infection in a very different way. In the sooty mangabey monkey, for example, the immune system does not suppress viral load, which stays high, yet the animal does not become ill. From the abstract:

“… Surprisingly we have found that SIV-infected sooty mangabey monkeys do not develop AIDS despite high level virus replication, short longevity of infected cells and limited anti-SIV specific cellular immune responses…

“Interestingly, an attenuated host immune response to the infection is manifest from early times during primary infection, suggesting that sooty mangabey evolution has selected for a limited, rather than an aggressive, host response. In all, these data suggest that the direct consequences of high level virus replication alone cannot account for the progressive CD4+ T cell depletion leading to AIDS, and that active antiviral cellular immune responses may not always be beneficial.

“Indeed, SIV-infected sooty mangabeys may be spared by their failure to mount significant antiviral immune responses, much of the indirect bystander damage seen in pathogenic primate lentivirus infections that both contributes to accelerated CD4 depletion and compromises host immune regenerative capacity. In contrast, following zoonotic transmission of SIV to non-natural hosts, the generation of active but incompletely effective immune responses may indirectly both increase the magnitude of overall T cell destruction and reduce the host immune regenerative capacity, thereby leading to the development of progressive immune deficiency as T cells lost to cumulative direct and indirect consequences of virus infection are not replaced.”

How to proceed

A treatment that prevents AIDS by reducing damage from HIV might be hard to recognise. It might not decrease viral load at all, or even increase it. The ultimate proof would be that people would not get sick over a long period of time. But it would probably be impossible to conduct clinical trials in the straightforward way — randomly assigning patients to antiretrovirals with or without the new treatment — because the effectiveness of antiretrovirals has made it almost impossible to run clinical-endpoint trials. Instead, new drugs today are approved by their effect on viral load, an endpoint that would not work in this case. (In fact, if an existing drug for some other medical purpose happened to work this way and prevent the development of AIDS without lowering viral load, we would probably not know it, even if many patients with HIV had used the drug coincidentally.)

How then might it be possible to get a handle on the development of this kind of drug? Here are some possible approaches:

Many patients have “discordant” viral load and CD4 counts. Instead of fitting the usual pattern of having higher CD4 counts if they have low viral load or vice versa, they either have a high CD4 count despite a high viral load, or a low CD4 count even though their viral load is low.

These two kinds of discordant patients could be compared to each other, to look for differences in how they respond to HIV infection. What could be learned from patients who can tolerate a high viral load and still maintain a high CD4 count — especially those who remained healthy despite having the high viral load for a long time? If the mechanisms could be identified, perhaps some kind of pharmaceutical intervention could help other patients do likewise.

If there are some patients who, like the sooty mangabeys, are long-term non-progressors despite having a high viral load, we probably would not have recognised them. Instead we would have treated their viral load, and attributed non-progression to the treatment. But these patients might be identified by careful examination of their medical records.

Basic research could look for the mechanisms involved, either in sooty mangabeys, other animals, or in any humans known to tolerate high HIV viral loads and remain healthy.

Of course differences in the virus as well as the host could be responsible for reduced disease progression despite high viral load. But still the host somehow avoids disease even though the virus reproduces well and does cause disease in non-native hosts.

For many years some physicians and researchers were interested in immune suppressive drugs to treat HIV. Their experience should be reviewed — especially since sooty mangabeys seem to have evolved an effective defence against AIDS that includes a notably unaggressive immune response. Existing drugs may be too non-selective or have too many side effects for widespread use. More selective immune-suppressive drugs could be developed.

Once a candidate harm-reduction drug is identified, it could be tested to see if it improves the health of patients who cannot control the virus with any existing treatment. Since their viral load cannot be controlled in any case, an experimental treatment to reduce viral damage could ethically be tested while the viral load stays high (necessary to see if the new treatment worked). A placebo control would be used since there is no approved HIV harm-reduction treatment. The volunteers could either take antiretrovirals or not as they chose. The study could look for T cell count increases, reduction of symptoms believed to be caused by the high viral load, and/or other evidence of clinical improvement. Such endpoints could show significant change quickly in a small number of patients (unlike the “clinical endpoint” of disease progression, which requires hundreds if not thousands of volunteers because it counts low-probability, all-or-nothing events instead of measuring continuous data on everybody).

A treatment that prevented viral damage without reducing viral load would not have the public-health advantage of antiretroviral treatment in making patients less likely to transmit the virus to others. But in practice, this kind of treatment would probably be combined with antiretrovirals for maximum benefit, so the risk of transmission would still be reduced.

A possible advantage of HIV harm reduction is that HIV develops resistance to all known antiretrovirals — and to the body’s immune responses as well. But a harm-reduction treatment would create different evolutionary incentives, as HIV variants would not need to evade either the therapy or the body’s defences in order to survive. They could do best by not provoking the immune system. And in the sooty mangabey example the viral load does not increase without limit until it kills the animal; there is still a setpoint, still a limit, and the animal remains healthy. So a harm-reduction treatment may also allow relatively harmless viruses (which would have an advantage here) to help crowd out more dangerous ones.

Perhaps such reasons explain why animals apparently evolved a strategy of maintaining health by preventing harm, even from continuous high levels of viruses still able to cause disease in other species. Human long-term non-progressors (at least those who have been identified) use a different strategy, of aggressive immune defence that keeps viral replication low enough to greatly delay escape from immune control. It seems likely that the former approach is the better one for controlling a virus that can mutate so rapidly. Possibly some patients are already benefiting from it, but under current medical and research practices we do not see them. For where viral load testing is available, treatment is available too, and almost no one gets viral load tests repeatedly unless they plan to treat a high viral load. Usually antiretroviral treatment would reduce the viral load and be credited for non-progression. And experimental HIV therapies that fail to lower viral load are not studied today.

As a result, a new kind of potential treatment for AIDS may have been overlooked.

Ref:
Feinberg M. Ignorance is bliss: how the natural hosts for SIV infection remain healthy despite long-term, high-level virus replication. Journal of Human Virology, 2002; volume 5, number 1, abstract #8.

Source: AIDS Treatment News, Issue #384, October 18, 2002

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