The 24th International AIDS Conference (AIDS 2022) in Montreal heard on Friday that for the first time scientists have developed a sensitive gene assay that can specifically find the tiny subset of ‘reservoir’ cells that harbour silent HIV infection by using nanotechnology to detect their distinct genetic signature.
“The holy grail of HIV cure research is to find a biomarker for the reservoir,” Professor Sharon Lewin, president-elect of the International AIDS Society told a press conference. This study is an important step towards finding such a biomarker, she said.
The reservoir problem
Dr Eli Boritz of the Vaccine Research Center in the US National Institute of Allergy and Infectious Diseases told the conference that what makes HIV infection lifelong is that the virus hides where it can’t be seen by the immune system – by inserting its genes into our own DNA inside a small minority of the cells that make up our immune system.
In untreated infection, HIV proliferates throughout the immune system, commandeering its T-cells to make new viral particles and in the process inflicting fatal damage on the immune system by killing off the CD4 T-cells, which co-ordinate cellular immunity. But a small amount of HIV gets sequestered inside inactive T-cells, which send out no signals of its presence.
Antiretroviral therapy (ART) stops viral proliferation but can’t get at the hidden viral DNA or kill off the cells that contain it. They persist – ready to activate and start turning out new viruses if ART is stopped.
A key component of most HIV cure strategies therefore involves finding these reservoir cells and either killing them off with supercharged immune therapies, or silencing them forever.
The problem is that we know the reservoir exists. But we don’t know which cells make it up, where they are, and whether they’re different in different people. We don’t have tools that can reliably identify them – and thereby target them with therapies. We don’t know whether they have special characteristics that make them go into a quiescent state and hide the virus, or whether they are especially receptive to HIV in the first place. And we don’t how to selectively reactivate them in the presence of ART, or how they reactivate and how to stop them, in its absence.
This invisibility of the reservoir cells makes curing HIV even more difficult than using similar cell-targeting therapies to cure cancer. Cancer cells do display lots of epitopes – the characteristic cell markers that alert an immune response – and so can be targeted by immunotherapies. With HIV, this is a much harder task.
"We don’t know which cells make up the reservoir, where they are, and whether they’re different in different people."
There are characteristics of cells that make them more likely to be, or to become, reservoir cells. For instance, there’s a cell-surface molecule called CD27. Its presence enables T-cells to become immunologically active and ‘remember’ the characteristics of viruses, while its absence on similar cells denotes that they have done the job and become quiescent. So, 90% of integrated HIV DNA resides inside CD27-positive cells. But most of this is degraded and mutated in the process of integration. The majority of intact HIV DNA – DNA that is capable of making entire new viruses – is inside CD27-negative cells, because they have the unusual characteristic of shutting off as soon as they are infected and thus perpetuating their infection.
The same applies to other cellular markers. Reservoir cells tend to be enriched in the CXCR4 co-receptor of HIV infection, as this is the one that enables infection with T-tropic virus – the type that tends to arise in chronic infection. And reservoir cells, not surprisingly, display increased sensitivity to HIV compared with other cells if they are woken up from their quiescent state.
The problem is that reservoir cells hosting intact viral DNA are very rare – sometimes only one in a million T-cells – so the majority of cells with these reservoir-cell characteristics won’t, in fact, be part of the HIV reservoir.
The only way to detect reservoir cells is to detect their hidden HIV DNA directly. To, quite literally, shine a light on it.
The FIND-Seq assay
The FIND-Seq assay involves several steps, each involving transporting and sorting individual cells through tiny channels considerably thinner than a hair.
T-cells are taken from the blood of HIV-positive people on ART, enclosed within individual ‘nano-capsules’ consisting of oil droplets with a gel skin, and transported along the tubes.
The memory subset of T-cells is first separated using their cell-surface characteristics. They are then ‘interrogated’ – stimulated so they produce lengths of RNA unique to their individual owner’s genome. Re-encapsulated, they are then passed through a laser beam whose signal alerts the system to lengths of unexpected viral DNA and sends the cells containing them to separate lab dishes.
Using this technique, the researchers first constructed a general map of which genes were unusually active or inactive. They compared genes within cells that did contain intact HIV DNA, versus ones that did not. Most ‘HIV-positive’ T-memory cells had quite distinct genetic signatures from ones that did not contain HIV. In particular, a lot of genes that were active in the cells lacking HIV were turned off in the cells that did have it. These included genes regulating the programmed death of cells and ones stopping them from proliferating. This indicated that these cells might have the properties that suited HIV latency.
But a subset of cells had overall genetic activity that did not correlate with the presence or absence of HIV. So the researchers then performed an iterative set of experiments to find if particular combinations or ‘modules’ of genes correlated with cellular HIV infection.
They found two, one consisting of 60 genes and one consisting of 85 genes – out of thousands of genes first inspected – that correlated strongly with intact HIV infection.
Narrowing these down again, they found four genes associated with HIV infection that governed the downregulation of gene transcription – in other words, genes that told other genes to turn off. Five others preserved cells in an inactive state, ensuring they did not too easily start differentiating, which would shorten their lives.
Three were responsible for turning off genes once a cell had done some genetic work – suggesting that these were the exact ones that turned cells into reservoir cells once they had been through the process of HIV integration. These genes exist primarily to protect cells from viral-induced toxic processes that might damage them. In other words, HIV may take advantage of the very genetic processes that have been developed to limit viral damage. And several other genes directly contributed to repairing nuclear and mitochondrial DNA that gets frayed and damaged – which is exactly what happens in viral integration.
What this all means, said Dr Boritz, is that we finally have a way of inducing reservoir cells to reveal their characteristic genetic signatures; that we know now, in detail, what those genetic signatures look like; and that those genetic signatures are exactly what you’d expect.
This implies that therapies designed both to ‘wake up’ reservoir cells and to target them for specific, self-induced cell death may in the future be much more exactly targeted. This could even lead to ways of stopping HIV establishing a reservoir and becoming a lifelong infection in the first place.
At present this is all lab-dish science. But we finally have an assay that may enable us to target HIV cure and immunomodulatory therapies much better.
Boritz E et al. Transcriptional programs of HIV silencing and cell survival in HIV-infected memory CD4 T cells under antiretroviral therapy. 24th International AIDS Conference, Montreal, abstract OALBX0102, 2022.