Could we eliminate HIV from the body altogether? Keith Alcorn looks at the latest milestones in a long journey of discovery.
Back in 1996, when highly active antiretroviral therapy (HAART) began to transform HIV care, there was much talk of its potential to eradicate HIV from the body altogether, which would amount to a cure.
I remember a standing-room only talk where combination therapy pioneer Dr David Ho, explained to the Vancouver International AIDS Conference how the new viral load test could measure the decline in virus levels in newly infected patients treated with HAART. The two-phase decline would probably last from one to four months, until all the cells presumed to harbour HIV had died off. Eradication looked entirely feasible from the optimistic viewpoint of 1996.
Little more than a year later, doubts began to accumulate. HIV was found to infect a group of longer-lived immune system cells called memory CD4 lymphocytes. By late 1999, scientists had concluded that it would take at least 60 years for this group of cells to die out. The idea of cure disappeared from the conference circuit.
Nonetheless, knowledge about the mechanisms that allow HIV to persist in the human body continued to accumulate. A number of tenacious scientists are still probing the reservoir of HIV-infected cells and learning more about how the virus can lurk silently within the genetic code of a few thousand human cells, ready to spark an explosion of viral replication if HAART ceases.
Suddenly, in 2008, scientists began discussing the subject with more enthusiasm. The field was partly stimulated into action by excitement over the apparent potency of the new integrase inhibitor raltegravir (Isentress), and a belief that its ability to clear virus more rapidly than other drugs might portend some greater capacity to penetrate into places other drugs could not reach. It was also nurtured by funding from the American Foundation for AIDS Research (amfAR), which never lost faith in the possibility of a cure for HIV.
Last November I attended a meeting in Washington where 40 scientists, convened by amfAR and the Treatment Action Group, sat down to thrash out the key scientific questions about how to cure HIV infection. A sign of the change in the scientific weather was a ten-minute appearance by Dr Anthony Fauci, the director of the US National Institute of Allergy and Infectious Diseases, an organisation with a budget of $4.7 billion to spend in 2009. Fauci’s appearance signalled that research into a cure was now being taken more seriously.
More research on possible ways of curing HIV infection was published this year alone than in the whole of the previous decade. In March a group of US researchers published a call for a global public-private partnership to accelerate research into a cure, and called on the US National Institutes of Health – the main funding body for medical research in the US – to devote long-term funding to the goal.
Why are scientists suddenly so interested, and what are the realistic prospects of a cure?
The Berlin patient
A key development which has got people talking about a cure again is the report of an apparent cure in a Berlin man who underwent a bone marrow transplant for treatment of leukaemia while also receiving antiretroviral drugs (ARVs).1 The man received bone marrow from a donor who had natural resistance to HIV infection; this was due to a genetic profile which led to the CCR5 co-receptor being absent from his cells. The most common variety of HIV uses CCR5 as its ‘docking station’, attaching to it in order to enter and infect CD4 cells, and people with this mutation are almost completely protected against infection.
More than two years after two bone marrow transplants (the first failed), the man remained free of HIV as far as the most sensitive tests could determine. Although the researchers who reported the case decline to describe it as eradication of HIV, the results are intriguing.
More research on possible ways of curing HIV infection was published in 2009 alone than in the whole of the previous decade.
However, the mechanism that has led to this long-term halt in viral replication (we’ll come to definitions of a cure later) is still unclear.
In bone marrow transplants the patient’s population of cancerous immune cells is destroyed and then the immune system repopulated by stem cells from the donor’s bone marrow. Did this eliminate HIV? Unlikely, because there have been previous cases of viral resurgence in people who received transplants.
Was it because the stem cells lacked the CCR5 receptor, leaving HIV no opportunity to enter new cells and replicate? Possibly, but most people have some viruses which are able to use other receptors, and in this patient, a tiny number were present before the transplant. Furthermore, some immune cells with CCR5 receptors were still present in the patient’s gut six months after transplantation.
Was it the use of immunosuppressive drugs after the transplant, which would stop HIV-infected cells ‘waking up’? It’s impossible to tell, although the key drug used (cyclosporine) has been tested as an adjunct to HAART to limit the number of target cells the virus might infect.
Despite these unanswered questions, researchers believe that the findings suggest a role for suppressing the production of CCR5-bearing cells, either through such transplants, or by gene therapy.
Scientists were sufficiently intrigued that they met in Berlin earlier this year to discuss how they could co-ordinate efforts to identify CCR5-lacking donors and expand the supply of stem cells from them, for example through sampling blood cells from the umbilical cord of babies born to mothers who have the mutation, in order to eventually facilitate stem-cell therapy.2
Gene therapy techniques which can transform stem cells – and all their descendents – into cells resistant to HIV entry may be a more practical option than looking for matching donors. Several US research groups announced in late October that they had received funding to explore techniques for engineering and introducing CCR5-deficient stem cells. If these approaches prove successful they will be expensive, so in the early stages it is likely that they would be reserved for people with no remaining treatment options or a cancer requiring bone marrow or stem cell transfer. But given that the lifetime cost of treating someone with HIV with ARVs is close to £200,000 if they live for at least 35 years after diagnosis,3 these techniques may become more cost-effective in time.
Other researchers are beginning to think in terms of combinations of drug therapies and immunological therapies that can rid the body of HIV-infected cells.
Challenge one – bringing HIV out of hiding
HIV infection is currently impossible to cure because when the virus infects an immune system cell, its genetic material becomes integrated into the human DNA in the cell. If the cell is activated to respond to an infection, it begins pumping out new viruses. These infect other activated cells in the vicinity, and the same cycle begins all over again.
As we have said, a population of HIV-infected cells persists in the body, ready to begin producing HIV when the viral sequence within it becomes activated. In these latently infected cells HIV is tucked away in a section of the genome not activated by normal cellular processes, rather like a moth chrysalis secreted in the corner of a cupboard, invisible to the naked eye. Only when it receives a specific chemical signal will it emerge.
Such latent virus is invisible to the immune system and therefore impossible to eradicate. But latency might be overcome by giving immune system cells a massive shock, causing them to pump out virus which can then be cleared by antiretroviral drugs, and so permanently reducing the number of infected cells.
This is a potentially risky strategy. The consequences of activating all the cells of the immune system potentially infected with HIV might be catastrophic, as researchers found in a recent trial of a drug being developed to treat rheumatoid arthritis. It was meant to modestly stimulate CD4 memory cells, but it had such an overstimulating effect six men suffered severe organ damage.
Studies have looked at several immune activation approaches in combination with HAART, but no study so far has demonstrated long-term reductions in the amount of cells containing HIV DNA.
Latent sequences are kept tucked up within neatly wrapped bundles of DNA, like cotton round a spool. The ‘spools’ are proteins called histones, and the DNA – with its secret payload of viral genes - can be kept from producing new proteins by the presence of enzymes called histone deacetylases (HDACs).
We have reached the theoretical limit of antiretroviral therapy. Robert Siliciano, John Hopkins University
A number of research groups have been investigating HDAC inhibitors, which would allow the spools to uncoil, as a means of switching on HIV and making it a target for destruction, and several drug companies are beginning to take an interest in this area.
An Italian research group used HDAC inhibitors in combination with a substance that would lower levels of the protective antioxidant glutathione in latently infected cells. This stresses the cell into greater HIV transcription when HDAC inhibition takes place. The stressed cell eventually self-destructs. The researchers dubbed the process, which has been tested so far only in the laboratory, ‘shock and kill’.4
HDACs regulate many vital cellular processes, and interfering with them could produce long-term side-effects unless the compounds chosen are highly specific for the HDACs involved in controlling HIV latency. For this reason, products already in use for other diseases, such as cancers, are likely to be tested in humans with HIV before novel products are used. A group from the University of Carolina recently reported that not all HDAC inhibitors stimulated viral replication to the same extent and we will need ones that maximise viral transcription while minimising toxicity.
Robert Siliciano of Johns Hopkins University and colleagues recently announced that they have developed a fast-throughput means of screening compounds to identify agents that can activate latent HIV without activating the CD4 cell.
Challenge two – finding the hiding places
Another key challenge is finding out where all these latently infected cells are hiding.
Intensifying treatment with new classes of drugs like integrase inhibitors has not proved capable of further reducing the very low levels of virus seen in people on HAART, suggesting that the virus is being released from places not affected by HAART – the so-called ‘reservoirs’.5,6
Robert Siliciano tested patients using ultrasensitive assays and has found that most people with a viral load below 50 copies/ml still have some detectable viral replication.
"We have reached the theoretical limit of antiretroviral therapy," he said at the 16th Conference on Retroviruses and Opportunistic Infections this year.
Any residual virus detected in a fully adherent person on HAART – including viral blips when levels can rise above 50 copies/ml – appears to be coming from latently infected cells in a reservoir that is invulnerable to HAART. One of these may be HIV-infected cells in the gut, which CD4 cells come into contact with during their normal trafficking around the body.
HIV infects cells in the lymphoid tissues, which are marshalling yards where foreign matter is presented to the immune system for inspection. Lymph nodes, which are full of CD4 cells, are distributed all around the body, and rapidly become packed with HIV after infection. However, the greatest concentration of cells infected with HIV lies in the abundant lymphoid tissue in the walls of the gut, which is the main route for foreign matter entering the body.
There is also speculation that the virus infects cells in the bone marrow that eventually differentiate into various immune system cells. Every time the cell divides, it takes with it a copy of the instructions for making HIV.7 This is certainly what happens after HIV infects memory CD4 cells, which retain information about previously encountered infections so that they can quickly respond if it is encountered again. These cells replicate more frequently as the CD4 count falls, perhaps explaining why the reservoir of HIV-infected cells is so much larger in people with chronic HIV infection.8
The reservoirs become established in the first weeks of infection, before replication is checked by the partially successful response of the immune system. However, the size of the reservoir is strongly influenced by the duration of unchecked viral replication and the severity of CD4 decline.9
Experiments by Anthony Fauci’s research group indicate that in a person who has received fully suppressive HAART for several years, the reservoir varies from one infected CD4 cell in every billion in people treated soon after infection, to one infected cell in every 10,000 CD4 cells in people who began treatment when chronically infected.
In almost all people who started HAART less than six months after infection, infectious virus could no longer be cultured from their cells after one year of treatment whereas it was still detectable in everyone who started treatment later, despite three to six years of HAART.10
“Antiretroviral therapy probably completely stops the entry of newly infected cells into this pool [the reservoir]. Our job is to make the exit of cells from this pool quicker, in a way that’s clinically practical and safe,” says Professor David Margolis of the University of North Carolina.
In people with good immune reconstitution, most of the integrated HIV genes are found in central memory cells. Clearing the reservoirs will require the elimination of these cells, in a very targeted way instead of blasting the immune system away with crude chemotherapy.
Challenge three – understanding how eradication happens
It will be challenging to test for residual virus, especially latent, replication-competent virus, and to be able to measure what’s going on when drugs are used against it.
Describing experiments to clear latently infected cells, David Margolis says that currently, “we’re doing something at one end [putting in drugs] and measuring something at the other end [observing changes in viral load and the number of infected cells], and what’s in between is a black box.”
What’s not clear is whether all latently infected cells are the same. Some researchers suggest that latently infected cells have a variety of behaviours, requiring a variety of drug targeting methods, and that some may be permanently stuck in a resting state, unlikely to be activated. In addition, some of the HIV genetic material is just junk - ‘replication incompetent’. But determining what’s what will require years of work to examine the latently infected cells.
The road ahead
The best chance of eradication, most experts agree, is likely to be in people who can be treated within weeks of becoming infected, before the reservoirs have a chance to become well-established.
In this patient group, Professor Routy suggests, we could use CCR5 inhibitors to limit the number of cells the virus enters, integrase inhibitors to prevent it installing its genes in the DNA of cells it does enter, and the cytokine interleukin-7 to activate cells that are infected in order to mobilise lymphocytes to kill cells that are producing HIV.
In people with chronic infection the biggest challenge is the size of the reservoir of infected cells. If that could be at least reduced, viral replication might be held at bay by immune responses.
Immune responses could be strengthened by therapeutic vaccination. The idea of therapeutic vaccination is to enhance the body’s own immune response to HIV by finding ways to hyper-sensitise parts of the immune system to viral components, with the intention of inducing an immune state that can keep viral load low without drugs, thereby prolonging the time that can be spent off HAART.
So far, though, this approach has produced little in the way of immune control of HIV, with the exception of preliminary results from a recent Canadian study which suggested that a vaccine tailored for each individual in the study appeared to keep HIV below pre-treatment levels during a 12-week treatment interruption.11 The ultimate aim of a therapeutic vaccine is to ensure that the viral load remains undetectable and that the cytotoxic T-cells, or CD8 cells, recognise and kill any infected cell that is on the verge of spewing out virus. Clearly there’s a long way to go.
The alternative is to look for strategies that can sustain latency, by further protecting HIV from activation and by exquisite targeting of latently infected cells. However, this is no more than a concept at present, with no hard evidence to show whether compounds already exist that can reinforce the latent state or induce a permanently ‘stuck’ state in the cells infected with HIV, rendering them unable to be activated and thus incapable of producing HIV with the ability to infect new target cells.
What sort of cure might be possible?
The preferred outcome must be complete eradication - a treatment programme that results in elimination of all traces of HIV from the body. This may prove very difficult.
A more plausible outcome is remission, the lack of detectable virus in the absence of treatment. Given the difficulty of finding and measuring HIV in latently infected cells, this may be the best that can be achieved.
In hepatitis C, cure is defined as a sustained virological response to treatment – no detectable hepatitis C viral RNA in the blood six months after treatment ends. However, there is evidence that very low levels of replication-competent hepatitis C viruses can persist in the blood for up to five years after successful treatment. Because hepatitis C does not become part of our own cells’ genes, it is easier to imagine eventually deleting every single bit of virus in the body.
Such data show how difficult the idea of eradicating HIV is, but do suggest that a treatment that mobilises the natural immune response to viral infection - alpha-interferon in the case of hepatitis C – may be crucial if infection is to be cured.
Even inducing an immune state that completely suppressed viral replication might not be enough. What if persistent viral replication continues in the brain, a ‘sanctuary site’ that drugs and the immune system may not completely be able to reach? And what if age-related loss of immunity eventually undermines remission?
For all these reasons, our ultimate goal may still need to be the elimination from the body of every cell containing potentially reproducible HIV. Research so far has indicated which roads might reach this goal, but travelling along them will be a long journey.
References
1. Hutter G et al. Long-term control of HIV by CCR5 delta32/delta32 stem-cell transplantation. N Engl J Med. 360: 692-8, 2009.
2. Hutter G et al. Transplantation of selected or transgenic blood stem cells – a future treatment for HIV/AIDS. J Int AIDS Soc 12: 10, 2009.
3. Devine A et al. Estimating the costs of HIV treatment in the UK. Proceedings of the Health Protection 2009 conference; 14–16 September 2009, University of Warwick, UK
4. Savarino A et al. “Shock and kill” effects of class-1-selective histone deacetylase inhibitors in combination with the glutathione synthesis inhibitor buthionine sulfoxamine in cell line models for HIV-1 quiescence. Retrovirology 6: 52, 2009.
5. Jones J et al. No decrease in residual viremia during raltegravir intensification in patients on standard ART. Sixteenth Conference on Retroviruses and Opportunistic Infections, Montreal, abstract 423b, 2009.
6. Hatano H et al. Evidence of persistent low-level viremia in long-term HAART-suppressed individuals. Sixteenth Conference on Retroviruses and Opportunistic Infections, Montreal, abstract 425, 2009.
7. Stebbing J et al. Where does HIV live? N Engl J Med 350: 1872-1880, 2009.
8. Chomont N HIV reservoir size and persistence are driven by T cell survival and homeostatic proliferation. Nature Medicine, June 2009.
9. ibid.
10. Strain MC et al. Effect of treatment, during primary Infection, on establishment and clearance of cellular reservoirs of HIV-1. J Infect Dis 191: 1410-8, 2005.
11. Routy JP et al. Safety and viral load changes in HIV-1 infected subjects treated with autologous dendritic immune therapy following ART. AIDS Vaccine 2009, abstract OA04-05, 2009.