Towards an HIV cure: Early developments reported at IAS, part 2
1 October 2012. Related: Conference reports, Cure research, World AIDS 19 Washington 2012.
Muirgen stack, HIV i-Base
Part one of this article was published in July/August HTB.
The announcement of the IAS Towards an HIV Cure scientific strategy at the 3rd IAS pre-conference symposium, held this year from 20-21 July, gave a new emphasis to advances in the field of cure research. 
The strategy outlines seven important areas of research where advances needed to be made if a cure (functional or sterilising) is to be realised:
- Why does HIV persist: immune and viral factors
- Where does HIV persist: tissue and cell reservoirs
- Immune activation and dysfunction on ART
- Natural models of HIV/SIV control
- How to measure persistent infection
- How to reverse latency: treatments
- Immune approaches, gene therapy, vaccine
Fortunately, some promising cure-related research was presented at AIDS 2012 – something that is hopefully sustained and expanded upon at further meetings.
Novel therapeutic approaches
A greater understanding of viral latency and persistence alongside an expansion of existing therapies is only likely to be one part of any future HIV cure (see Part 1 of this report published in the previous issue of HTB). The creation of novel treatments may be just as essential if targeting viral production and/or replication in sanctuary and/or reservoir sites.
Carolina Garrido and colleagues from UNC Chapel Hill, North Carolina presented a poster on using gold nanoparticles to target viral reservoirs in the brain. 
Early research from this group showed that fluorescently labelled (TAMRA) gold nanoparticles successfully enter lymphocytes, macrophages, astrocytes and human brain microvascular endothelial cells (HBMECs) after 24-hours incubation. FACS analysis showed up to 15% of CD4+ T-cells contained TAMRA. Moreover, the ability of the nanoparticles to cross the blood-brain-barrier was confirmed in vivo by intravenous tail injection in mice. Mouse brain gold content reached up to 869 ppb/gram of tissue, and was also observed by microscopy. The nanoparticles were then conjugated to a raltegravir derivative to assess antiviral activity. This reduced viral replication to 25–38% by day 5 compared to 100% HIV replication in media alone. Unfortunately, this was not compared to an unconjugated raltegravir derivative or other ARVs. Nevertheless, these preliminary findings showed initial efficacy and targeting specificity for nanomedicine-based approaches to HIV treatment – although any implications for accumulating gold nanoparticles in brain tissue with chronic treatment were not addressed. 
Frauke Christ from the University of Leuven, Belgium, presented pre-clinical data on a new class of integration inhibitors, known as LEDGINs.  LEDGINs bind allosterically to a site on integrase where the cellular co-factor LEDGF/p75 attaches. LEDGF/p75 acts to anchor the viral DNA to the host chromatin, before integration. LEDGINs have previously been shown to inhibit HIV replication in vitro via this mechanism. 
In the current study, the researchers evaluated the ability of LEDGINs to block the catalytic activity of integrase directly, reporting further inhibition of HIV replication in MT2 and PBMC cells, as well as activity over a broad range of viral clades, and even with viruses containing mutations conferring resistance towards other integrase inhibitors.
Although still in their developmental infancy, there at least appears possible future use for LEDGINs – either on their own or concomitantly with other integrase inhibitors. However, whether they will retain their pre-clinical efficacy in animal and human clinical trials remains to be seen.
Targeting the integrated virus and immunotherapies
Helga Hofmann-Sieber from the Heinrich Pette Institute, Hamburg, presented research, which directly addressed an HIV cure (functional or sterilising) by creating a system that can remove integrated HIV proviral DNA from infected human cell cultures.  This approach uses a viral LTR-specific recombinase (Tre-recombinase) that the researchers have previously shown to excise HIV-1 proviral DNA from infected human cell cultures. Tre-recombinase is an enzyme based on the bacterial cre-recombinase, which removes sections of DNA that are flanked by a specific series of nucleotides called loxP. Unfortunately HIV proviral DNA does not contain any loxP sequences. So, the researchers chose a mutant cre-recombinase that could recognise a sequence of nucleotides in the LTRs of integrated HIV.
Applying the technique to HIV positive humanised mice meant creating an advanced lentiviral self-inactivating (SIN) vector that expressed Tre-recombinase conditionally in HIV-infected cells. Both human CD4+ T-cells and CD34+ haematopoietic stem cells (HSC) were successfully transduced prior to engraftment in HIV-1 positive Rag2-/-gammac-/- mice. The pronounced antiviral activity via the Tre-recombinase system was not associated with undesired cytopathic effects in the transduced cells from Tre-recombinase over expression.
Excising integrated HIV-1 DNA would theoretically allow for the eradication of HIV-1 from the body. However, transducing all latently infected cells with Tre recombinase in the many viral reservoirs found in the body remains infeasible. Therefore, the Tre system needs to be modified to allow easier administration before it can be considered for clinical trials like other “anti-latency” drugs such as the histone deacetylase (HDAC) inhibitor vorinostat.  First however, the researchers must address a question raised at the end of the presentation which highlighted the Tre-recomninase system to be dependent on the transcription of the viral protein TAT. Unfortunately, disruption of the expression of TAT is a marker of latency, meaning that the Tre-recombinase system would have minimal efficacy on excising proviral DNA from latently infected cells.  However, this does make the Tre-recombinase system redundant and perhaps by pairing with other equally novel lentiviral vectors that deliberately over stimulate TAT, the system could continue to be advanced. 
Shifting away from novel molecular therapeutics, Lydia Trautmann from VGTI, Florida gave an interesting presentation on possible advances in immunotherapies.  She focused on better characterising the differences between HIV-specific CD8+ T cells before and during ART. Her findings included that under low HIV antigen loads (caused by viral suppression from ART), the dominant HIV-specific CD8+ T cell clonotype gained poly-functionality – recognising more epitopes of HIV antigens. This led to a small but efficient clonotype developing under ART. Dr Trautmann concluded that if ways were found to expand these high-affinity HIV-specific CD8+ T cells in vivo, they might be able to control the virus without ART.
Fortunately, an oral abstract by Scott Kitchen from the UCLA AIDS Institute, Los Angeles covered this topic.  After recognising the cytotoxic CD8+ T lymphocyte (CTL) response as a critical component in controlling HIV infection, the researchers set out to enhance and expand CTLs’ effects in vivo. They utilised molecularly cloned HIV-specific T-cell receptors (TCRs) derived from CD8+ T-cells, which were then used to genetically transduce human haematopoietic stem cells (HSCs). The transduced cells were then introduced into a humanised mouse and were allowed to differentiate into mature human CD8+ CTLs. Mice expressing the transgenic HIV-specific TCRs were compared to control mice after both were infected with HIV-1.
They observed successful differentiation into mature CTLs and migration into multiple anatomic sites. They also crucially saw significant reduction in plasma HIV RNA levels, which correlated with both levels of reconstitution with cells bearing the HIV-specific TCR and antigen-driven T cell expansion.
These early findings from a humanised mouse model demonstrated the importance of the CTL response and how it can be expanded and improved. By relying on the CTLs own ability to transverse to multiple compartments in the body, it could also potentially override some of the difficulty ARVs may have in reaching sanctuary sites.
Stem cell transplantation
Taking host immune system modification even further, Timothy Henrich from Brigham and Women’s Hospital, Boston presented two case reports of allogeneic stem cell transplantation in two HIV positive individuals.  Both had haematological malignancies and had previously undergone autologous stem cell transplant. The patients remained on ART and were given a reduced-intensity conditioning (RIC) form of allogeneic stem cell transplant. Immunosuppressive therapy was eventually given in the form of prednisone or tacrolimus/sirolimus to treat chronic graft-versus-host disease (GVHD) after the transplant.
Total HIV-1 DNA was still seen up to 2 months after transplant in patient A (87 copies/million PBMCs) and up to 3 months after transplant in patient B (281 copies/million PBMCs). However after 8 and 9 months respectively, total HIV-1 DNA and 2-LTR HIV-1 DNA (copies/million PBMCs) became undetectable. This was also true for plasma HIV-1 RNA, which due to the sensitivity of the assay can only be shown to be <3 copies/ml.
Parallels will naturally be drawn to the “Berlin patient” (Timothy Brown) who received a stem cell transplant from a donor homozygous for the CCR5 delta-32 mutation.  However, the stem cells received by both patients A and B were from donors with normal CCR5 expression. Whether, this undermines the CCR5 delta-32 mutation being necessary as a mechanism to explain Timothy Brown being labelled as “cured” is unclear.
The authors proposed multiple possibilities to explain why HIV could not be detected; namely, GVHD and/or the effect of cytotoxic therapies given to treat it. However, a fuller understanding will have to wait until further samples are analysed to see if any HIV can be detected. More information can be found in a recent interview with the co-researcher Dan Kuritzkes from Harvard Medical School, Boston where he hints at the protective role of ART – acting as “PrEP on a cellular level”. 
It is clear that cure research covers many different areas and that any likely successes may involve a combination therapy approach. These are tentative stages of cure science, but there are still reasons for optimism. The most obvious being the open nature of cure research, which stresses the importance of collaboration between scientists in the field and is endorsed by the “Towards an HIV cure” working group. This leaves an update of cure-related advances at the 20th international AIDS conference (AIDS 2014) in Melbourne, Australia as an appealing prospect. 
Unless stated otherwise, all references are to the Programme and Abstracts for the 19th International AAIDS conference 22-27 July 2012, Washington.
- International AIDS Society (IAS). Towards an HIV Cure: Global Scientific Strategy. July 2012
- Garrido C et al. Gold nanoparticles to improve drug delivery to the central nervous system: targeting HIV reservoirs in the brain. Poster exhibition THPE014.
- Alkilany A and Murphy C. Toxicity and cellular uptake of gold nanoparticles: what we have learned so far? J Nanopart Res (September 2010) 12 (7): 2313-2333.
- Christ F et al. Pre-clinical evaluation of HIV replication inhibitors that target the HIV-integrase-LEDGF/p75 interaction. Oral abstract TUAA0301.
- Christ F et al. Rational design of small-molecule inhibitors of the LEDGF/p75-integrase interaction and HIV replication. Nat Chem Biol (June 2010) 6 (6):442-448.
- Hofmann-Sieber H et al. Towards HIV eradication: excision of HIV-1 proviral DNA by Tre-recombinase in HIV-positive humanized mice. Oral abstract TUAA0302.
- Archin N et al. Administration of vorinostat disrupts HIV-1 latency in patients on antiretroviral therapy. Nature (26 July 2012) 487: 482-485.
- Karn J. The molecular biology of HIV latency: breaking and restoring the Tat-dependent transcriptional circuit. Curr Opin HIV AIDS (January 2011) 6 (1):4-11.
- Macias D et al. A lentiviral vector that activates latent human immunodeficiency virus-1 proviruses by the overexpression of tat and that kills the infected cells. Hum Gene Ther (November 2009) 20 (11):1259-1268.
- Trautmann L. Novel approaches: treatment and HIV pathogenesis. Immunopathogenesis and its treatment. Symposia Session. 23 July 2012, 2.30-4.00pm, Washington.
- Kitchen S et al. In vivo suppression of HIV antigen specific T cells derived from engineered hematopoietic stem cells. Oral abstract TUAA0303.
- Henrich T et al. Long-term reduction in peripheral blood HIV-1 reservoirs following reduced-intensity conditioning allogeneic stem cell transplantation in two HIV-positive individuals. Oral abstract THAA0101.
- Allers K et al. Evidence for the cure of HIV infection by CCR5delta32/dellta32 stem cell transplantation. Blood (10 March 2011) 117 (10):2791-2799.
- A Barton. Paths to a cure: Dr. Dan Kuritzkes discusses experiences of two patients. 16 August 2012.
- The 20th International AIDS conference 20-25 July 2014 Melbourne, Australia.