Cure Research Takes Center Stage: Proof of Concept for Activating the Latent Reservoir
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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. 
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.  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.  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]
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  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. 
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. 
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. 
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.  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. 
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". 
This article was provided by HIV i-Base. It is a part of the publication HIV Treatment Bulletin. Visit HIV i-Base's website to find out more about their activities, publications and services.
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