Targets proliferate in HIV cure research

Many approaches described at this year's symposium
Dr Daniel Kuritzkes of Harvard Medical School gives keynote address: Progress and challenges in HIV cure research. Image by: Marcus Rose/IAS.
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The Towards an HIV Cure two-day symposium has become a fixture in advance of the International AIDS Society conferences and this one featured a more varied range of experimental approaches than ever in the search for ways of eliminating HIV infection from the body.

Dr Daniel Kuritzkes of Harvard Medical School, in his opening talk, told the delegates that to some extent the proliferation of different approaches was due to early disappointments in the cure field. We still only have one person, Timothy Ray Brown, who has been cured of HIV and six other cancer patients in whom the same stem-cell transplant therapy had been tried had all died – a reminder that a procedure as exacting as a bone-marrow transplant is never going to be an approach that can be used generally.

The main approach that cure researchers are still working on is the so-called ‘Shock and Kill’ strategy. This uses immune stimulants to induce the cells in which dormant HIV lies hidden – the so-called reservoir cells – to come out of hiding. The hope is then that their activation will in itself lead to their death through natural immune exhaustion; if not, the aim is to target them with directed cell-killing drugs. Without eliminating this reservoir, a small minority of cells capable of spitting out new HIV will remain in the body; experiments have shown that HIV can reappear even when undetectable with the most sensitive viral load tests, as in the case of the ‘Mississippi Baby’.

Glossary

cure

To eliminate a disease or a condition in an individual, or to fully restore health. A cure for HIV infection is one of the ultimate long-term goals of research today. It refers to a strategy or strategies that would eliminate HIV from a person’s body, or permanently control the virus and render it unable to cause disease. A ‘sterilising’ cure would completely eliminate the virus. A ‘functional’ cure would suppress HIV viral load, keeping it below the level of detection without the use of ART. The virus would not be eliminated from the body but would be effectively controlled and prevented from causing any illness. 

reservoir

The ‘HIV reservoir’ is a group of cells that are infected with HIV but have not produced new HIV (latent stage of infection) for many months or years. Latent HIV reservoirs are established during the earliest stage of HIV infection. Although antiretroviral therapy can reduce the level of HIV in the blood to an undetectable level, latent reservoirs of HIV continue to survive (a phenomenon called residual inflammation). Latently infected cells may be reawakened to begin actively reproducing HIV virions if antiretroviral therapy is stopped. 

immune system

The body's mechanisms for fighting infections and eradicating dysfunctional cells.

protein

A substance which forms the structure of most cells and enzymes.

receptor

In cell biology, a structure on the surface of a cell (or inside a cell) that selectively receives and binds to a specific substance. There are many receptors. CD4 T cells are called that way because they have a protein called CD4 on their surface. Before entering (infecting) a CD4 T cell (that will become a “host” cell), HIV binds to the CD4 receptor and its coreceptor. 

Since the ‘shock and kill’ strategy has gained wide currency, there have been disappointments: the experimental agents used to reverse so-called ‘latency’ have certainly stimulated virus production by cells - but without resulting in any decrease in the size of the viral reservoir. This appears to be because the drugs chosen - HDAC inhibitors like panobinostat or romidepsin – have other, unforeseen immune effects, including suppressing activity in the very CD8 cells that might be central to the ‘kill’ part of the process.

Nonetheless, Kuritzkes said, for the time being “latency reversal is a necessary, if not sufficient condition in reducing the reservoir of HIV-infected cells”.

He told aidsmap.com: “Most of the interventions that are likely to eliminate infected cells require that the virus is visible to the immune system. The alternative idea, that of permanently suppressing viral production by reservoir cells” (as in the study published last week of a tat inhibitor) “at the moment would seem to involve taking a latency suppressor pill every day instead of antiretroviral therapy. That’s not really a cure.”

Post-treatment controllers

Other cure research has involved investigating immune responses both in spontaneous elite controllers of HIV – who maintain low viral loads and high CD4 counts from the start of their infection – and the so-called ‘post-treatment controllers’ like members of the VISCONTI cohort, who start HIV therapy usually early, but later manage long periods off treatment. These cases are fascinating to researchers as they mimic the ‘functional cure’ of HIV that is one of the goals of cure research. The cure symposium heard about another case – see separate report – this time of a young woman now aged 18 who has maintained an undetectable viral load off therapy for twelve years.

By definition, however, most people with HIV cannot become post-treatment controllers as, in general, controllers are people who start treatment very early, and even then probably have to develop a very specific immune response to HIV – one possibly easier to induce with a preventative vaccine than with post-infection treatments.

Preventing latency

For these reasons, while there were presentations of romidepsin and of other drugs designed to reverse latency such as ingenol, a relative of the latency-reverser prostratin, there was more excitement at this symposium about drugs that aimed to prevent cells ever becoming quiescent and entering latency.

This would involve inhibiting the activity of latency promoters such as the cellular protein PD-1, which ‘shuts down’ activated T-cells, thus making them invisible to the immune system.  PD-1 is not the only latency promoter, and Colleen McGary of Emory University proposed using drugs that block both PD-1 and another latency promoter called CTLA-4 (CD152) to prevent latency, since cells that express both receptors are for more efficient at incorporating HIV DNA than ones that espress one or neither. One CTLA-4 blocker, ipilimumab, is already used for melanoma. However PD-1 in particular is a ubiquitous part of the immune system and drugs that block its activity have proved to be rather toxic when used in cancer research.

Another cellular target is a cellular protein called SAMHDI which is already targeted by an existing anti-leukaemia drug called dasatinib. Jose Alaci of the Instituto de Salud Carlos III in Spain told the symposium that SAMDHI reduces cell division and in T-cells reduces their ability to produce new virus; however it usually operates only for a short part of the immune cycle and is then phosphorylated, which means it becomes inert. Dasatinib stops this phosphorylation and maintains the activity of SAMHDI, which means that it stops viral transcription happening inside the cell, meaning that it could act like a reverse transcriptase inhibitor, but one that acted on the cellular response to HIV.

Kill – picking off infected cells with antibodies…

As well as preventing cells entering latency, we also need better ways for the immune system to recognise and kill off the HIV-infected reservoir cells that are forced out of hiding. Only a tiny minority of reservoir cells ever wake up and start producing whole, replication-competent virus in what are called ‘reactivation events’: one poster at the cure symposium from researchers at the University of New South Wales estimates that there is on average one ‘reactivation event’ in the reservoir of HIV-infected cells every five to eight days, though under effective antiretroviral therapy these never become fully-fledged infection events that result in detectable viremia. Each reactivation event is literally started off by one cell: the researchers told aidsmap that the average length of time an individual reservoir cell would remain quiescent before it had a 50% chance of reactivating was around 17,000 years.

Marcus Altfeld of Harvard Medical School gave an interesting presentation where he described the different stages of reproduction of HIV that newly-stimulated reservoir cells go through. As we said, fully-fledged activation is quite rare: cells actually sending out viral particles can be targeted with passive immunisation with monoclonal antibodies, and John Mascola of the US National Institutes of Health said that antibodies that treat skin cancer had already been devised that have a half-life in the blood of over six months.

This would not be a cure, of course: to maintain antibody levels in the blood would require a therapeutic vaccine that stimulated the production of neutralising antibodies and the problem with antibodies to HIV is that they have always been specific to only one strain of HIV. Mascola said that the most broadly neutralising antibodies were now capable of destroying 90% of viral strains or tagging for destruction the cells that bear them. While allowing 10% of strains to escape would not be enough for a cure, he described the development of new ‘bispecific’ antibodies which could combine two, each with 90% neutralisation capacity, on one molecule or could combine a viral neutraliser with an antibody that attracted CD8 cells to come and attack the infected cell.

….with CD8 cells…

More of the reservoir cells produce viral proteins or lengths of viral RNA without ever assembling complete viruses. They would nonetheless display ‘epitopes’ or short samples of viral proteins on their surface that act as distress signals and attract the CD8 cells that destroy infected cells. Again, the problem with CD8 cell response to HIV, as with antibody response, is that the virus can mutate to escape it. But a broad-spectrum therapeutic vaccine given after a highly-suppressive antiretroviral therapy regimen that has driven viral load down as far as possible could, it is hoped, detect cells preparing for reactivation events so efficiently that they would be caught before HIV has a chance to escape their immune surveillance. Sarah Fidler of the UK cure research consortium CHERUB described a trial of one in which two different CD8-inducing vaccines are given 24 and 32 weeks after the initiation of a highly-suppressive four-drug therapy.  Another vaccine that could be useful in this context is the CMV-based vaccine that caused a stir last year with its apparent ability to bring about a functional or even complete cure in monkeys, and phase I trials of this are underway in humans.

Another way of avoiding viral escape from immune surveillance is to elicit an immune reaction to those parts of the virus that are tightly ‘conserved’ – areas that the virus cannot mutate without crippling itself. In HIV these areas are usually kept hidden well away from the immune system. When HIV attaches to the CD4 molecule on cells, however, its gp160 protein that does the job of attaching to CD4 has to change shape to expose a highly-conserved part of the gp41 fusion protein for a very short time. The broadly neutralising antibodies have ‘prongs’ that can reach in and attach to this conserved area, and in the early days of AIDS, soluble CD4 protein was used experimentally in a BID to force this ‘conformational change’ in gp160. But Andres Finzi of McGill University described a new small-molecule CD4 analogue called JP-III-48 which, unlike soluble CD4, could be an oral pill. This would ‘jam open’ the gp160 molecule and expose the conserved sections to patrolling CD8 cells, thus further shrinking the size of the reservoir.

…and with natural killer cells

The hardest challenge is to find ways of directing the attention of the immune system towards cells at the very earliest stage of a reactivation event. At this point they are starting to transcribe integrated proviral DNA into lengths of RNA that get chopped up (‘spliced’). These spliced RNAs then serve as the templates for individual viral components. People with cells that contain large amounts of unspliced HIV RNA are less likely to experience reactivation events than people whose cells contain spliced DNA. As the HIV protein tat is absolutely essential in the process that splices DNA, the announcement last week of a small-molecule tat inhibitor was particularly interesting.

However this is not the only HIV protein that could be targeted to prevent reservoir cells getting beyond this phase. Cells just starting to produce foreign RNA do display ‘stress proteins’ on their surface that, while not clearly foreign and therefore attracting a cellular immune response, do attract the attention of the innate immune system – the evolutionarily oldest, least selective but fastest response force in the immune system. These stress proteins bind to a cellular receptor called NKG2D that is displayed by natural killer cells, the shock troops of the innate immune system. HIV however has a protein called nef, one of whose jobs is to repress the expression of the stress proteins that attract the natural killer cells. A small-molecule nef inhibitor or an anti-nef vaccine could block the activity of nef, increase the expression of cellular stress proteins, and generate an innate immune response capable of attacking reservoir cells at a very early stage of their activity.

Making cells look immune to HIV

Lastly, there is the option of using gene therapy to try to repopulate the immune system with cells that lack the HIV receptors necessary for replication, and particularly the CCR5 receptor used by most transmitted viruses. This was of course the basis for Timothy Ray Brown’s cure: his new bone marrow cells came from someone who naturally lacked CCR5. Approaches using patient’s own CD4 cells that have their receptor genes removed and are then re-infused back into the patient, without taking the drastic step of deleting their entire immune system first and were among the first cure experiments conducted in humans. Matt Sharp, one of the first volunteers in such a trial, was at the cure workshop and described the enduring benefits – few side effects, and a lengthy CD4 boost.

One of the problems with deleting the CCR5 receptor in immune cells is that HIV can use another receptor, CXCR4 and indeed CXCR4-using virus has appeared in at least one of the failed experiments that tried to reproduce the Timothy Ray Brown cure. Unfortunately, while people without CCR5 can stay in good health, CXCR4 is a much more essential part of the immune system and CXCR4 blockers used as HIV treatments have failed due to unacceptable toxicity. However a team at the University of Pennsylvania have managed to engineer CD4 cells that carry on their surfaces a ‘fused peptide’ which is a glued-together combination of part of the HIV gp41 fusion protein with either the human CXCR4 or CCR5 receptors. Cells expressing this fused peptide ‘look as if’ they lack the CCR5 or CXCR4 receptors to the immune system and are therefore resistant to HIV infection.

Maintaining the HIV cure as a priority

This is only a small sample of numerous cure strategies and drug targets explored during the two-day cure workshop. There were two other therapeutic vaccine experiments conducted in monkeys, both producing promising immune changes. There were very early investigations of the role of gut microbes in inducing cellular vulnerability to infection and latency. There were various other promising drug and vaccine targets described.

What there was not this year was any one significant breakthrough or research direction: cure research is at the stage of following many promising leads but lacks a strong signal that would indicate which, of a variety of strategies, is the one to pursue. In all likelihood, as Marcus Altfeld reminded the symposium, it will take a combination of approaches. Matt Sharp told the symposium that now was “not the time to rush to judgement on any individual cure approach”.

He added that it was essential to include the search for a cure in any discussion of “ending AIDS”. He was concerned, he said, that the recent conversations about treatment access, treatment as prevention and PrEP and targets such as 90/90/90 had not mentioned curing HIV and he did not want to see it dropping off the agenda or off the list of funders’ priorities. While developments in the reduction of HIV incidence seen in places as diverse as San Francisco and Botswana were welcome, he added, only curing as well as treating and preventing HIV would truly bring an end to the epidemic.

“How can we end AIDS, without a cure?” he asked.    

References

There are too many individual studies to cite here but all presentations made at the 2015 Cure Symposium will be uploaded to their site at http://www.iasociety.org/What-we-do/Towards-an-HIV-Cure/Events/2015-Symposium.

See http://www.iasociety.org/Web/WebContent/File/HIV_Cure_Symposium_Programme_2015.pdf for the symposium programme.