Simon Collins, HIV i-Base

For the last two years the major HIV conferences, including CROI and the International AIDS Society (IAS) have included cure research prominently in the main programme. This is new and significant.

At CROI in 2010, Anthony Fauci, head of the US National Institute of Allergy and Infectious Diseases (NIAID) announced that the US government would be launching new funding for cure research. [1]

Many of the researchers in this field have been working on a cure years, some for decades. But the new drive for this research to receive better funding is clearly an important factor in how quickly future progress will be made.

The new funding may, in part at least, have also been driven by the responsibility that America has assumed as the largest donor for global HIV treatment programmes. Over the last ten years, ARV access in low and middle-income countries has increased from less than 0.5 million people in 2002 to over 6.5 million people in 2012. A long-term alternative to lifelong treatment is therefore likely to be an economic as well as a medical necessity. While current cure research uses specialised and expensive procedures, as with all new developments, including ARVs, high initial costs would hopefully be driven down to become more widely affordable.

The IAS has also been developing a leadership role to coordinate global funding for cure studies and to hopefully focus on a research map that will minimise duplication. [2] The IAS organised workshops prior to each of it’s last two conferences and another is planned prior to the Washington meeting in July 2012. [3] Several community workshops, including one before CROI this year have also contributed to broadening awareness of the potential for a cure. [4, 5]

In addition to an oral abstract session this year, CROI included several helpful presentations of the current research in the preconference workshops for young investigators, particularly the overview by John Mellors and the talk on animal models for latency by Vincente Planelles. [6, 7]

The Berlin cure

Whether through mediated immunity (referred to as a functional cure) or eradication (a sterilising cure), the ability to overcome lifelong treatment has always been an ultimate goal, even while the focus for recent years shifted to achieving more effective, tolerable and durable treatments.

The first report of a cure following stem cell transplantation from a donor who was naturally resistant to HIV infection (he was homozygous for the delta-32 deletion in CCR5) was at CROI in 2008 [8] and increasing press coverage since had made this a highly publicised case, and brought optimism to cure research.

The mechanism responsible for curing Timothy Brown (a.k.a. the Berlin Patient) who has been off treatment now with no evidence of HIV for over four years has not been isolated to a single component from a complex and risky set of procedures.

In addition to myeloablative chemotherapy and total body irradiation to kill both HIV infected and uninfected immune cells, he received antithymocyte globulins, cylcosporin, mycophenolate acid (MMF) and gemtuzumab (anti-CD33) that would also have killed HIV-infected and uninfected cells, followed by allogeneic stem cell transplants from a donor homozygous for delta-32 mutation, which should have reseeded an immune system resistant to CCR5 HIV infection, he developed graft vs host disease (GVHD) indicating he had accepted the donor immune system. These procedures have a 25% mortality risk and he underwent each procedure twice as the course was repeated.

An oral presentation at CROI reported on ten patients on suppressed ART who underwent autologous (self-donated) hematopoietic stem cell transplantation for AIDS related lymphoma, which is a less risky procedure than that used by Tim Brown. Unfortunately, persistent HIV viraemia was still detected in 9 of 10 patients post-transplant, with a median viral load of 1.5 copies/mL (range: <0.2 to 26) and median total HIV-1 DNA of 554 copies/million PBMCs (range: <0.4 to 2179). 2-LTR circles were detectable post-transplant in only 2 of 10 patients (range: 1 to 7 copies/million PBMC). The only patient with undetectable plasma viral load had the highest levels of HIV-1 DNA and 2-LTR circles. Additionally, plasma viraemia persisted in a patient with undetectable HIV-1 DNA in PBMC. Although the authors concluded that this showed that the CCR5 delta-32 donor was essential in the Berlin case, patients in their study also did not have total body irradiation, graft vs. host disease, and were reinfused with their own stem cells, which could have included HIV-infected T-cells. [9]

A further US study is about to open of allogeneic stem cell transplant in HIV positive people with bone marrow failure with the hope that 1 or 2 of the 15 patients may also be able to be matched to a delta-32 donor to see if the Berlin case can be repeated. [10]

This will involve overcoming the difficulty of finding and matching a delta-32 donor who is also compatible on 8 HLA types. At the community cure meeting prior to CROI, John Zaia from the City of Hope Cancer Centre near Los Angeles described an initiative to develop an inventory

of cord blood stem cell donors as an international resource, and to date, out of 13,000 donors tested, 90 have been identified as being homozygous for the CCR5 delta-32 deletion. [5]

First activation of latently infected resting T-cell reservoir in vivo

While many aspects of this research are controversial, there is broad consensus on the need for a strategy to overcome the reservoir of long- lived, latently infected, resting CD4 cells that harbour integrated HIV and that are not reached by current ART.

Most notably, an oral presentation at CROI included results from a proof of concept study that viral latency might be overcome. David Margolis from the University of North Carolina presented results in an oral late breaker presentation that the use of a single dose of the histone deacetylase (HDAC) inhibitor vorinostat (suberoylanilide hydroxamic acid, SAHA) is able to activate latently infected resting CD4 cells. [11] In 2005, Margolis presented results from using another HDAC inhibitor, valproic acid, to stimulate the latent reservoir.

Of the 11 human histone deacetylase, HDACs 1, 2, and 3 are the primary enzymes that limit activation of HIV integrated into cells by producing a barrier that maintains latency. Vorinostat is a selective inhibitor of HDAC 1, 2, and 3 that has been shown to induce HIV expression from latently infected resting cells ex vivo. However, vorinostat, although approved as a cancer treatment also has mutogenic properties.

In this proof of concept study, the change in the latent reservoir was determined by measuring cell associated HIV RNA specifically in the resting cell population. This involves harvesting approximately four billion lymphocytes from each aviraemic patient by leukopheresis that are treated with magnetic antibody beads to leave 200-1000 resting CD4 cells that can be tested by RNA PCR.

Six study participants had baseline measures of activation, that were tested ex vivo after exposure to vorinostat and that demonstrated that a change was measurable in all patients. Each patient also undertook a single 200 mg safety dose and a separate single 400 mg dose of vorinostat for a PK study to decide the timing for the second leukopheresis used to determine efficacy.

Following a second, therapeutic 400 mg dose, all six patients responded with a highly significant mean 4.8 fold increase (range 1.5-10-fold) of RNA expression in resting CD4 cells (p<0.01). The treatment was well tolerated with no reported side effects associated with vorinostat and none greater than grade 1. Of note, and perhaps surprisingly, no increases in HIV plasma RNA were detected using a single copy/mL test.

The study concluded that is the first demonstration of activation of latent resting HIV-infected CD4 cells in vivo. However, these results are still preliminary. While the proof-of-concept is exciting, Margolis suggested that this might be seen as the equivalent of a “ddC moment in relation to HAART”.

Additionally, other molecules may be more effective compounds to activate latency and in vitro data suggesting panobinostat as more active that vorinostat were presented in a poster. [12]

Earlier in the same conference session Liang Shan reported that latently infected resting CD4 cells treated with vorinostat survived despite viral cytopathic effects, even in the presence of autologous CD8 cells from most patients on ART concluding “that stimulating HIV-1-specific CTL responses prior to reactivating latent HIV-1 may be essential for successful eradication efforts and should be considered in future clinical trials”. [13]

Treatment during early infection

Theoretically, the easiest targets for cure research might be those patients diagnosed earliest in their infection, who promptly start treatment and who maintain suppressed viraemia for many years.

Although the latent cell reservoir is established within weeks of infection and is likely to be slowly reduced after years on effective ART, in nearly all patients, viraemia rapidly returns within weeks if treatment is interrupted. Even when HIV is reduced to being present in less than 1 in 1.7 billion cells, this is sufficient for systemic infection to quickly be reestablished (within two months) if treatment is stopped. [14]

While levels this low might question the importance of a treatment to target the viral reservoir, they can so far only be achieved with very early treatment and/or many years of viral suppression. The need to reduce the viral reservoir more quickly will be a concern for everyone else who started ART during chronic infection.

Rapid viral rebound without treatment has been widely reported in numerous treatment interruption studies. However, several small cohorts have also reported viral control in a minority of patients, usually in those who initiated treatment in acute infection and maintained undetectable viral load for several years.

Last year at CROI, the ANRS Visconti study reported small numbers of patients who started treatment in early infection (after serconversion, median viral load >100,000 copies/mL), maintained viral suppression for >3 years on treatment and who have subsequently controlled viraemia off treatment for >6 years. [15] This year at CROI similar cases were reported in posters by two other groups.

Maria Salgado and colleagues reported a single case of a patient who initiated treatment during seroconversion (viral load >750,000 c/mL, western blot indeterminate) for three years and after stopping ART has since maintained viral load suppressed to <50 copies/mL off-treatment for more than nine years. Initial and current viral isolates are dual CCR5/CXCR4 tropic and fully replication-competent in vitro. Minimal viral evolution has been detected over the 11 years.

He is reported to currently have low titers of neutralising antibodies to heterologous and autologous HIV-1 isolates, and his CD8+ T cells do not have potent HIV suppressive activity suggesting a mechanism other than CTL-mediated suppression reported in elite controllers. [16]

Alain Lafeuillade from General Hospital, Toulon (who is also one of the key organisers of the International HIV Persistence Workshop that has been meeting every two years since 2003) reported that 17% (8/45) of a cohort of patients treated at seroconversion for a median of 2.2 years (range 1.8 to 4.0) have remained off treatment for more than 10 years, two of whom remain suppressed to <20 copies/mL (median 2,500 copies/mL for the other six). The 37 people who restarted treatment (due to confirmed CD4 decline to <350 cells/mm3) did this after a

median of 5.0 years (range 3.0-8.0) off-treatment. The study suggested the protective mechanism could be relate to early ART reducing the HIV reservoir but also emphasised that such responses seem to be rare. [17]

A poster from Joseph Margolick and colleagues reported small differences in viraemia between people diagnosed in early infection (within a year of infection) and randomised to immediate treatment (n=57) year) and those who did not start early treatment (n=24). However, study numbers were very low at the evaluation point (24 months after stopping treatment of 24 months after diagnosis) due to ~20% loss to follow- up and exclusion of people who restarted treatment for other reasons. [18]

Generally small differences were also reported from early treatment in the larger SPARTAC study that randomised almost 400 people (diagnosed within 6 months of infection) to deferred ART or immediate treatment for either 3 months or 12 months, and who then stopped treatment. [19]

However, in the context of eradication research, two oral presentations suggested that early treatment, while too late to prevent the establishment of the viral reservoir, might reduce the pool of latently infected cells.

Maria Buzon and colleagues estimated the size of the viral reservoir in patients treated for more than ten years who initiated ART within 3 months of infection (n=9) and compared levels integrated and total HIV DNA levels to people who started treatment during chronic infection (n=26) and to elite controllers (n=37). [20]

Integrated and total DNA levels were significantly lower in both primary treated (p=0.06 and p=0.001, respectively) and elite controllers (p=0.003 and p<0.0001, respectively) compared to those treated in chronic infection. In addition, the ratio between total and integrated HIV-DNA was significantly lower in early treated and elite controllers (both p=0.04 vs chronic) with no differences between acute and EC groups.

Although patient numbers were small, differences were also reported when comparing how soon treatment had been started with patients treated during Fiebig stage III or IV vs stage V having significantly lower levels of both total and integrated HIV DNA after two years.

An oral presentation by Alan Perelson from the Los Alamos National Laboratory used mathematical modelling to look at the impact of early treatment of 27 people treated during acute infection on the size of the latent reservoir, and the relationship of both to initial viral load and target cell ability. [21]

This study also reported that earlier ART, including earlier during primary infection, had a measurable impact related to the initial size of the reservoir, with patients who already started with very low levels of resting cell infection (who also had low levels of peak viral load) experiencing less change in the reduction of resting cell decay. The model also suggested that CD4 T cell increases in response to successful ART was not increasing the viral reservoir.

Research into a functional cure

Other groups are focusing on immunological interventions that would support a functional rather than eradicating cure.

Pablo Tebas from the University of Pennsylvania, presented additional safety and efficacy results from the use of zinc finger nuclease (ZFN) modification of CD4 cells (using SB-738) to a CCR5-deleted phenotype (in development by Sangamo BioSciences). [22]

This process involves harvesting cells by apheresis, treating them with SB-738 to produce 13-35% of cells with CCR5-detetions in vitro. The cells are then expanded, cryopreserved and 5-30 billion cells are reinfused into the donor patient.

Results were combined from three studies: one in ART responders (baseline CD4 >450 cells/mm3) who subsequently interrupted treatment (group 1, n=6) and two in immune non-responders (baseline CD4 <500 cells/mm3) who have not interrupted treatment (group 2, combined n=15). Initial results from these studies were presented at CROI and ICAAC conferences last year.

Most patients were male, white, mean age 48, with a long history of HIV infection (median 12 and 18 years in group 1 and 2 respectively). Mean CD4 count and CD4:CD8 ratio were 921 (+222) cells/mm3 and 1.4 (+0.6) in group 1 and 335 (+89) cells/mm3 and 0.7 (+0.3) in group 2.

Duration of follow-up is now a mean 325 days (range 90 – 738 days).

After infusion, CD4 cells increased by about 1500 cells/mm3 in group 1 (n=6), these then decreased during the treatment interruption but which remained significantly above baseline during follow-up. CD4 responses in group 2 involved an increase of about 500 cells/mm3 which then dropped by about 200-300 which then remained stable out to over a year in the patients who did not interrupt treatment. The expansion of CD4 cells was associated with increases in IL-2, IL-7 and IL-15.

The CD4:CD8 ratio increased significantly in both groups, normalising and remaining at approximately 1.0 throughout follow-up in the group 1 and increasing to approximately 2.5 for the six patients in group 2 decreasing during the treatment interruptions but then remaining stable.

The modified cells continued to be detected through follow-up at 2% of circulating CD4 cells at 48 weeks for most patients. Levels were higher during the treatment interruption for group 2 and then dropping to 2%. Circulation of cells to other tissue sites was confirmed by multiple rectal biopsies where levels of the CCR5-modified cells were comparable to those in blood or higher throughout follow-up.

During the treatment interruption viral load rebounded over the first 8 weeks to around 100,000 copies/mL in a similar way to other interruption studies dropping by one log during the last 4 weeks off-treatment to levels that were generally higher than pre-ART. After three months, when treatment was restarted, viral load become undetectable again in all six patients. One person, later found to be heterozygous for the delta-32 mutation, had a lower rebound (to 10,000 c/mL) and then resuppressed viral load to undetectable by week 8 and remained undetectable off treatment until restarting as per protocol at week 12.

This group used a new method to measure changes in the viral reservoir based on levels of HIV DNA sensitive to low copy numbers (although unable to distinguish between integrated DNA and 2-LTR circles. They reported no detectable change in 4/6 patients with one person have a transient 4-fold increases at week 12 and 20 during the interruption but returning to baseline levels and one person experiencing a 9-fold increase that returned to baseline 16 weeks after restarting treatment.

Side effects were mild and transient, mainly within 24 hours of the infusion (mild chills, fever, headache, fatigue) but included one report of arthritis lasting a few days and abnormal garlic-like body odour.

Next steps include using immunomodulatory drugs such as cylcophosphamide to promote engraftment and increase the percentage of modified cells and studying other patients who are heterozygous for the delta-32 deletion.

Several studies presented studies where pegylated interferon (peg-IFN) was added to ART prior to stopping HIV treatment and continuing peg-IFN. The results suggested that viral rebound was delayed by the peg-IFN via an immune-mediated rather than antiviral mechanism, but these were small studies with short-term follow up (12 and 24 weeks). [23, 24, 25]

comment

The timeline for a cure at this meeting was optimistically referred to as being at least ten years. HIV is a tricky puzzle: the virus is resilient and the range of immune responses is complex. Nevertheless, these advancements in several key and linked areas are crucial advances.

Several networks are encouraging collaborative research in order to be able to compare and evaluate different approaches. [26, 27]

Numerous compounds that are already licensed are already being looked at for their potential to overcome latency. These include prostratin, lonomycin, thapsigargin (a calcium pump inhibitor), PMA, typhostin-A (a non-selective typhosin phosphate inhibitor), CD3/CD8 antibodies for TCR signaling, PLA, toll like receptor 7 (TLR7 including GS-9620) and protein kinase-C (PKC) agonists. Several companies including Gilead and Merck are already screening for and have identified other potential HDAC inhibitors.

The important of informed community participation in partners in this research is particularly important given the ethical considerations for study volunteers. If a mechanism is discovered to cure HIV more widely, it may still only work in some patients.

With current treatment able to nearly normalise life expectancy, a cure has a high bar to overcome. Some of this research will involve asking people on stable treatment to interrupt therapy and some of the interventions will have potentially greater toxicity than their current ART. At least for the foreseeable future, the potential risks in these initial studies are likely to outweigh any personal benefits.

Treatment during primary infection early treatment may put someone at a preferential state to respond to parts of a strategy to cure HIV, and some people diagnosed this early may want to take this decision. Because the latent pool is smaller in such patients, those initiating ART during acute HIV may be the best candidates for pilot studies attempting HIV eradication.

References

Unless stated otherwise, all references are to the Programme and Abstracts for the 19th Conference of Retroviruses and Opportunistic Infections, 5–8 March 2012, Seattle.

1. Fauci A. A Fauci. The HIV/AIDS research agenda: a view from NIAID. 17th CROI 2010, 16-19 February 2010, San Francisco. Plenary session 5. February 16, 2010.

http://retroconference.org/2010/data/files/webcast_2010.htm

2. International AIDS Society (IAS). Towards a cure: global scientific strategy. February 2011.

http://www.iasociety.org/Default.aspx?pageId=370

3. International AIDS Society (IAS). Towards a cure: global scientific strategy. Washington meeting 2012.

http://www.iasociety.org/Default.aspx?pageId=349

4. Treatment Action Group. Report: HIV cure-related clinical research workshop, October 2011.

http://www.treatmentactiongroup.org/cure/2011-workshop-report

5. ATAC Cure Research Workshop. 4 March 2012. 6. Mellors J. HIV Reservoirs and Cure Research. Workshop: Program Committee Workshop for New Investigators and Trainees (Part 2) Monday 5 March 2012 11:00 AM, Seattle.

http://retroconference.org/static/webcasts/2012/

7. Planelles V. Models for the Study of Latency. Workshop: Frontiers in Laboratory Science Workshop. Monday 5 March 2012 2:30 PM, Seattle.

http://retroconference.org/static/webcasts/2012/

8. Hutter G et al. Treatment of HIV-1 infection by allogeneic CCR5-D32/D32 stem cell transplantation: a promising approach. Poster abstract 719.

http://www.retroconference.org/2008/Abstracts/31704.htm

9. Cillo A et al. Plasma viremia and cellular HIV-1 DNA persist despite autologous hematopoietic stem cell transplantation for AIDS-related lymphoma. Oral abstract 154.

http://www.retroconference.org/2012b/Abstracts/42793.htm

10. Allogeneic Transplant in HIV Patients (BMT CTN 0903)

http://clinicaltrials.gov/ct2/show/NCT01410344

11. Margolis D et al. Administration of vorinostat disrupts HIV-1 latency in patients on ART. Oral late breaker 157LB.

http://www.retroconference.org/2012b/Abstracts/45315.htm

12. Rasmussen T et al. The histone deacetylase inhibitor (HDACi) panobinostat (LBH589) stimulates HIV-1 expression more potently than other HDACi in clinical use and disrupts HIV latency at clinically achievable concentrations. Poster abstract 370.

http://www.retroconference.org/2012b/Abstracts/43423.htm

13. Shan et al. Elimination of the latent reservoir for HIV-1 requires induction of cytolytic T lymphocyte responses. Oral abstract 153.

http://www.retroconference.org/2012b/Abstracts/43184.htm

14. Chun T-W et al. Rebound of plasma viremia following cessation of antiretroviral therapy despite profoundly low levels of HIV reservoir: implications for eradication. AIDS 2010: 24 (18): p 2803–2808 (27 November).

http://journals.lww.com/aidsonline/Fulltext/2010/11270/Rebound_of_plasma_viremia_following_cessation_of.6.aspx

15. Saez-Cirion A et al. Long-term HIV-1 Control after interruption of a treatment initiated at the time of primary infection is associated to low cell-associated HIV DNA levels: ANRS VISCONTI Study. Poster abstract 515.

http://www.retroconference.org/2011/Abstracts/41477.htm

16.Salgado M et al. 357 Title: Prolonged Control of Replication-competent Dual-tropic HIV-1 following Cessation of HAART. Poster abstract 357.

http://www.retroconference.org/2012b/Abstracts/43398.htm

17.Lafeuillade A et al. Long-term control of HIV reservoir after a 2-year ART course at acute infection. Poster abstract 358. http://www.retroconference.org/2012b/Abstracts/43198.htm http://www.retroconference.org/2012b/PDFs/358.pdf (PDF)

18. Margolick J et al. 356 Effect of randomized HAART on viral suppression off therapy in patients with acute/early HIV infection. Poster abstract 356.

http://www.retroconference.org/2012b/Abstracts/45103.htm

http://www.retroconference.org/2012b/PDFs/356.pdf (PDF)

19. Fidler S et al. The effect of short-course antiretroviral therapy in primary HIV infection: final results from an international randomised controlled trial; SPARTAC. 6th IAS Conference on HIV pathogenesis, treatment and prevention. 17-20 July 2011. Rome, Italy. Oral abstract WELBX06. See:

http://i-base.info/htb-south/1528/

20. Buzon M et al. Treatment of early HIV infection reduces viral reservoir to levels found in elite controllers. Oral abstract 151.

http://www.retroconference.org/2012b/Abstracts/44634.htm

21. Perelson et al. Immediate antiviral therapy restricts resting CD4+ infection but does not accelerate the decay of latent infection. Oral abstract 152.

http://www.retroconference.org/2012b/Abstracts/43222.htm

22.Tebas et al. Induction of acquired CCR5 deficiency with zinc finger nuclease-modified autologous CD4 T cells (SB-728-T) correlates with increases in CD4 count and effects on viral load in HIV-infected subjects. Oral abstract 155. http://www.retroconference.org/2012b/Abstracts/43132.htm

23.Mexas A et al. Concurrent Measurements of Total and Integrated HIV DNA Provide Insight into the Mechanism of Reduced Reservoir Size in an Interferon-alpha followed by Structured Treatment Interruption Trial. Poster abstract 374.

http://www.retroconference.org/2012b/Abstracts/44147.htm

24. Papasavvas E et al. Immune Correlates of Sustained IFN-alpha-mediated Suppression of HIV Replication: Association with IFN-alpha-mediated Signaling and Increased NK Cell Responses. Oral abstract 93.

http://www.retroconference.org/2012b/Abstracts/43948.htm

25. Azzon L et al. Pegylated interferon-alpha 2A monotherapy induces durable suppression of HIV-1 replication and decreased HIV DNA integration following ART interruption. Poster abstract 631.

http://www.retroconference.org/2012b/Abstracts/43413.htm

26. Collaboratory of AIDS Researchers for Eradication (CARE) and the Delaney AIDS Research Enterprise (DARE).

https://www.delaneycare.org/

27. Defeat HIV – The Delaney Cell and Genome Engineering Initiative, Fred Hutchinson Cancer Research Center, University of Washington, Seattle.

http://defeathiv.org

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.

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

In addition to the prevention studies and the progress on pipeline drugs that made most headlines (see the previous issue of HTB), a third set of presentations through the meeting supported the IAS Conference Statement on the need for the cure. [1]

That ‘the Cure’ might again re seen as an achievable goal for research was resuscitated in keynote lectures from NIAID lead Anthony Fauci several years ago and several medical networks, including the IAS, already hold annual meetings to coordinate different approaches. US public funding now requires cure research as a key work stream for HIV treatment networks.

Whilst the scientific challenge of curing HIV has been the consitent focus for many committed researchers, the renewed level of funding is clearly driven by the financial challenge of providing lifelong global treatment. Even when the generic costs are reduced to less than $100 per person per year, current treatment programmes need to more than double and then be sustained for coverage to meet the existing need. Perhaps increasing the resources for cure research is therefore perhaps also the most ethical way to be able to withdraw from responsibility for funding global treatment.

However, these sessions in Rome were mostly held in the smaller rooms, filled to capacity. They were also frustratingly insular with few being available as webcasts or slides to download, including the plenary session by IAS President Elect, Françoise Barré-Sinoussi. [2]

Neither the IAS pre-conference workshop (New concepts in HIV Immunopathogenesis, Treatment and Vaccine Strategies) nor the rapid summary report from that workshop in the main IAS conference by Nicolas Chomont was webcast. However, slides are available from some of the workshop sessions and for the summary by Chomont. [3, 4]

A satellite meeting cosponsored by amfAR and IAS also was not webcast, although the slide presentations can be downloaded. [5] This meeting focused on whether:

• viral replication persists on HARRT;
• eradication research can progress in animal models or is dependent on human studies;
• eradication is most likely to come from targeting the viral reservoir or more recent approaches using gene therapy.

Several other presentations at the conferences looked at strategies to selectively reactivate the reservoir of latently infected resting CD4 cells, either at the pre- or post-integration step. This challenge is highlighted by the pool being estimated to be less than one in a million resting cells for someone on stable treatment with undetectable viral load.

Some researchers believe that success in this goal might eradicate HIV, though this is dependent on whether current treatment suppresses replication sufficiently to halt viral evolution. This might turn out to be possible, as it has been the conclusion from several intensification studies that have shown no further reduction on low level viraemia after increasing the potency of a three drug combination with a fourth drug, including an integrase inhibitor. [6] An oral presentation from Brunetta and colleagues reported no impact on CD4 reservoirs in gut-associated lymphoid tissue obtained from sigmoid colon biopsies at 48 weeks of follow up following intensification with raltegravir. [7]

A case reported by Chun and colleagues in an article in AIDS last year perhaps also supports this view. [8] This paper described one person – ‘the Toronto patient’ – who was enrolled and treated prior to seroconversion. Viral load was suppressed to <50 copies/mL on HAART for more than ten years, driving the pool of infected CD4 T cells down to less than one in 1.7 billion cells. Against advice, the person decided to stop treatment under research conditions. Viral rebound only occurred after 50 days with an increased to 1600 copies/mL followed by spontaneous suppression by day 95 back to undetectable. Subsequently, viral load steadily increased to approximately 8600 copies/mL on day 143 when treatment was restarted.

So one interpretation of this case could be to emphasise the difficulty of eradication – even with such an early, effective and sustained level of treatment. Another interpretation is that eradication might almost have been achieved. Perhaps another month, or year, or few years on treatment might have been sufficient to final exhaust the remaining pool on resting infected cells. This study is unlikely to be repeated.

Another more optimistic, but also unexplained, set of cases includes the 32 patients from the ANRS Visconti study reported at CROI this year. This group received antiretroviral therapy within ten weeks of seroconversion for a median of three years (1-7.5 years). Five of these people sustained virological control for a median of 6 years (range 4-10) after treatment discontinuation. [9] It is unclear why similar cohorts (Rosenberg, Walker et al.) have not had the same success.

However, over time, so long as treatment is maintained, the resting pool of infected cells might be able to be agitated to become active, most likely by using multiple approaches. This could reduce the time needed to eliminate this reservoir from decades down to years – with residual virus mopped up by antiretrovirals, allowing treatment to be stopped.

Importantly, research into activation of latently infected cells is already investigating a broad group of drugs that are already licensed. Studying HIV transcription at the molecular level is driving the understanding of differences between latent and productively infected CD4 cells including HDAC-1 and methylation sites in latent infection with the hope that these targets might switch cells away from latency.

A comprehensive review of potential molecules by Sharon Lewin and Christine Rouzioux in the 24 April edition of AIDS [10] was the basis of one of the presentations at the IAS cure workshop. [11]

These include histone deacetylase (HDAC) inhibitors (vorinostat, romidepsin, panabinostat, entinostat, belinostat, givinostat and at least nine others), a methylation inhibitor (5-azacytidine), cytokines (IL-7 – Eramune group, IL-15) and an antialcoholic (disulfiram). Immune modulators with similar potential incude antibotics (minocycline), antirheumatics (auranofin), anti-PD-1 (MDX-1106) and protein kinase C modulators (bryostatins and others). Many of these compounds are already being studied in HIV-positive people.

An oral presentation by Claire Vandergeeten from the Vaccine and Gene Therapy Institute reported results from in vitro studies that suggest that IL-15 therapy may be used as a strategy to deplete the latent HIV reservoir while IL-7 maintains the reservoir both in vitro and in patients on stable HAART. [12]

This research is important and exciting. Many of these compounds have been studied for several years and for other studies are ongoing. A combination therapy approach is therefore likely to have a greater chance of success, for example, valproic acid or vorinostat plus prostratin. [13]

However, other researchers believe that an as yet unidentified sanctuary site would prevent the latent reservoir from being a slowly diminishing pool that theoretically might wear itself out, with or without stimulation to do so. This includes Steven Deeks at UCSF who co-chairs the IAS working group on cure research and was heads a recent $4 million grant from the US NIH to develop a strategy to eradicate HIV. [14]

This raises the importance of finding out whether any compartments are actively replenishing the viral reservoir, currently untouched by the maximal suppression measured by plasma viral load.

If this is the case, then any strategy to activate latently infected cells would not be successful. In November 2010, Yuki and colleagues reported that ongoing replication (measured by unspliced HIV RNA in CD4 T cells) is present in higher levels in gut sites (duodenum, terminal illeum, right colon and rectum) compared to that in PBMCs in patients on HAART with viral load suppressed to <40 copies/mL. [15]

The same group then showed that intensification with raltegravir in this group of patients produced a reduction in levels of unspliced RNA in the terminal ileum and a trend towards reduced T cell activation in other gut sites. [16]

For these researchers, looking for the impact of intensification studies in plasma viral load is searching in the wrong place. If tissue compartments are a source of ongoing viral replication, the spillover pool found in plasma may have limited relevance.  Other cellular sites distinct from CD4 T cells contributing to the latent cellular reservoir include macrophages, hematopoetic stem cells, naive T cells, astocytes, thymocytes and others.

Also, Maria Buzon and colleagues in Nature Medicine in 2010 reported increases in episomes following raltegravir intensification (shown by an increase in 2LTR in PMBCs) as evidence of de novo infection and reduced levels of activation. The extremely low level or replication is used to account for the non-development of resistance even over years. Also perhaps indicating that the chronic source for new virus drives a limited number of rounds of infection. [17]

Finally, in one of the few late breaker presentations with slides posted online, Hiroyu Hatano working with Deeks’ group at UCSF reported that viral persistence was consistently associated with markers of immune activation and dysfunction (including PD-1 expressing cells) rather than plasma viral load. These measures were particularly elevated in people on treatment with low CD4 counts despite treatment (less than 350 cells/mm3 compared to higher), suggesting that patients below this cut-off present a more difficult and sobering challenge to any approach to a cure.

More optimistically, Hatano noted that the preferential expression of PD-1 by latently infected cells supports targeting this molecule as a strategy for depleting HIV reservoirs. The AIDS Clinical Trials Group (ACTG) in the US is planning an exploratory trial of Merck’s experimental PD-1 inhibitor for this purpose. [18]

References:

Unless stated otherwise, all references are to the abstracts and conference programme of the 6th IAS Conference on HIV Pathogenesis, Treatment and Prevention, 1720 July 2011, Rome.

1. The Rome Statement for an HIV Cure: Major HIV/AIDS Stakeholders Call for HIV Cure Research to be Accelerated. International AIDS Society, July 2011.
   http://www.iasociety.org/Default.aspx?pageId=583
2. Barré-Sinoussi F. Discussing past and future accomplishments of HIV research. Abstract MOSS0103.
   http://pag.ias2011.org/session.aspx?s=83
3. Towards an HIV Cure: Insight into Residual Viral Replication, Establishment of Reservoirs and Understanding Mechanisms of Persistence. Conference workshop WEWS03.
   http://pag.ias2011.org/session.aspx?s=70
4. Chomont N. New concept in HIV: HIV immunopathogenesis, treatment and vaccine strategies – report back from pre-conference. Symposium WESY01.
   http://pag.ias2011.org/session.aspx?s=15
5. Controversies in HIV Cure Research. Joint IAS and amfAR workshop MOSA02.
   http://pag.ias2011.org/session.aspx?s=39
6. McMahon D et al. Short-course raltegravir intensification does not reduce persistent low-level viremia in patients with HIV-1 suppression during receipt of combination antiretroviral therapy. Clin Infect Dis. 2010 March 15; 50(6): 912–919.
   http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2897152/
7. Kovacs C et al. Effect of intensification of long-term highly active antiretroviral therapy (HAART) with raltegravir on proviral HIV-1 DNA in gut associated lymphoid tissue (GALT): a randomized, placebo controlled trial. 6th IAS, Rome 2001. Oral abstract MOAA0103.
   http://pag.ias2011.org/Abstracts.aspx?SID=53&AID=1432
8. Chun T-W et al. Rebound of plasma viremia following cessation of antiretroviral therapy despite profoundly low levels of HIV reservoir: implications for eradication. AIDS: 27 November 2010 – Volume 24 – Issue 18 – p 2803–2808.
   http://journals.lww.com/aidsonline/Fulltext/2010/11270/Rebound_of_plasma_viremia_following_cessation_of.6.aspx
9. Saez-Cirion A et al. Long-term HIV-1 control after interruption of a treatment initiated at the time of primary infection is associated to low cell-associated HIV DNA levels: ANRS VISCONTI study. 18th CROI 2011, Abstract 515.
   http://www.retroconference.org/2011/Abstracts/41477.htm
10. Lewin SR, Rouzioux C. HIV cure and eradication: how will we get from the laboratory to effective clinical trials? AIDS: 24 April 2011 – Volume 25 – Issue 7 – p 885-897.
    http://journals.lww.com/aidsonline/pages/articleviewer.aspx?year=2011&issue=04240&article=00001&type=Abstract
11. Lewin S. Contribution of the immune system to HIV persistence. Workshop: Towards an HIV Cure: insight into residual viral replication, establishment of reservoirs and understanding mechanisms of persistence, July 2011, Rome.
12. Vandergeeten C et al. Differential impact of IL-7 and IL-15 on HIV reservoir persistence. 6th IAS, Rome 2011. Oral abstract MOAA0101.
    http://pag.ias2011.org/Abstracts.aspx?SID=53&AID=2604
13. Reuse S et al, Synergistic activation of HIV-1 expression by deacetylase inhibitors and prostratin: implications for treatment of latent infection. PLoS ONE 4(6): e6093.
    http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0006093
14. NIH supports new research strategy for finding a cure for HIV. (July 2011).
    http://www.ucsf.edu/news/2011/07/10201/nih-supports-new-research-strategy-finding-cure-hiv
15. Yukl SA et al. Differences in HIV burden and immune activation within the gut of HIV-positive patients receiving suppressive antiretroviral therapy. Journal of Infectious Diseases. Published online 12 October. 2010;202:000-000. DOI: 10.1086/656722.
    http://jid.oxfordjournals.org/content/202/10/1553.full
16. Yukl SA et al. Effect of raltegravir-containing intensification on HIV burden and T-cell activation in multiple gut sites of HIV-positive adults on suppressive antiretroviral therapy. AIDS. 2010 Oct 23;24(16):2451-60.
    http://journals.lww.com/aidsonline/Abstract/2010/10230/Effect_of_raltegravir_containing_intensification.4.aspx
17. Buzón M et al. HIV-1 replication and immune dynamics are affected by raltegravir intensification of HAART-suppressed subjects. Nature Medicine 16, 460–465 (2010).
    http://www.nature.com/nm/journal/v16/n4/full/nm.2111.html
18. Hatano H et al. Cell-based measures of viral persistence are associated with immune activation and PD-1+-expressing CD4+ T cells. 6th IAS Conference, Rome 2011. Oral late breaker WELBA01.
    http://pag.ias2011.org/abstracts.aspx?aid=4801