‘Gentler cure’ technique produces delay in viral rebound in some people taken off HIV therapy

Pablo Tebas presenting at CROI 2019. Photo by Liz Highleyman.

Researchers have introduced genetic changes into human T-cells that make them virtually immune to HIV infection, and the viral load in HIV-positive people who are inoculated with these cells returns more slowly when they are taken off therapy, the recent Conference on Retroviruses and Opportunistic Infections (CROI 2019) heard.

The challenge now is to increase the proportion of cells in the body that have the immunity gene added. This might possibly result in a functional cure of HIV, with the virus unable replicate even in the absence of HIV drugs.

The top story at CROI 2019 was that a second person may have been cured of HIV infection. But most reports also emphasised that the bone marrow transplant procedure that achieved both this cure, and the previous one of Timothy Ray Brown 12 years ago, is dangerous, expensive and not something that would ever be attempted in a patient who did not have cancer.



A protein on the surface of certain immune system cells, including CD4 cells. CCR5 can act as a co-receptor (a second receptor binding site) for HIV when the virus enters a host cell. A CCR5 inhibitor is an antiretroviral medication that blocks the CCR5 co-receptor and prevents HIV from entering the cell.

analytical treatment interruption (ATI)

As part of a research study, when study participants are requested to interrupt their antiretroviral therapy (ART) and be closely monitored. Most of these studies are in the field that aims to eventually achieve ‘ART-free remission’ or ‘HIV cure’. They usually look at the time it takes for the viral load to rebound after the interruption, following which ART is restarted.


A unit of heredity, that determines a specific feature of the shape of a living organism. This genetic element is a sequence of DNA (or RNA, for viruses), located in a very specific place (locus) of a chromosome.


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. 


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. 

So can a similar feat be achieved more safely? Promising results in animal experiments produced monkeys that appear to have most of the HIV DNA removed from their cells.

But a second study, this time in humans, reproduced the genetic change that cured the two patients more exactly, and by means of a safe and repeatable technique. The two cured patients had their T-cells replaced with ones from donors with a genetic mutation called CCR5-delta 32 that means their cells don’t have the CCR5 receptor molecule on their surface that most strains of HIV need to attach to before they can infect a cell.

In the experiment presented by Dr Pablo Tebas of the University of Pennsylvania in Philadelphia, T-cells taken from 15 people with HIV were cultured in a lab dish with a gene-editing enzyme called a zinc finger nuclease (ZFN). This modified their CCR5 gene so it changed to the HIV-resistant variant. The cells were then reintroduced into the patients in what is called an autologous transplant – i.e. the modified transplant comes from the patient themselves, so there should be no problem with rejection.

This is not a new technology: ZFN enzymes were used to modify cells in experiments originally reported to CROI in 2011. In that experiment, a viral vector – the shell of a common-cold virus – was used to introduce the ZFN enzyme into cells. The reintroduced CCR5-negative cells initially made up about 22% of the T-cell population, but were replaced over time by the patients’ own CCR5-positive cells.

The current set of experiments, however, used a different technique called electroporation to get the ZFNs into cells. The problem with using viral vectors is that the cells develop immunity to them so they can only be used once. Electroporation can be used many times to create multiple cycles of modified cells that can be inoculated. In this technique, the T-cells are incubated alongside the ZFN enzymes in chambers in the presence of an electric current. This causes cell walls to become permeable and the ZFNs or other substances one wishes to introduce will diffuse into the cell.

As well as using a different and repeatable technique to create autologous CCR5-negative T-cells, the experiments explored a couple of other possibilities. Firstly, could the proportion of modified T-cells in the body be increased if a low dose of an immune-suppressant drug (cyclophosphamide) was used prior to their inoculation, to reduce the number of CCR5-positive cells in the body? And secondly, while only 1% of people of northern European ancestry have two copies of the CCR5-delta 32 gene, which means they are virtually immune to HIV, a larger proportion – up to 20% – have one copy of this gene (so-called heterozygosity). Would they have a better response?

There were 15 people in this study, in five groups of three. Three people received no cyclophosphamide. Six people – three heterozygous for CCR5 and three with no copies of the delta-32 mutation – received a lower dose of cyclophosphamide, and six people received a higher dose. The doses were not high enough to create significant toxicity and were about the same used to treat the autoimmune disease lupus, Dr Tebas said.

The creation of the CCR5-negative T-cells took about ten weeks, and the cyclophosphamide was given two days before the infusion of the cells. Eight weeks later the patients, who were all HIV-positive people with high CD4 counts (median CD4 count 831 copies/mm3), stopped their antiretroviral therapy (ART). The analytic treatment interruption (ATI) was for a fixed period of 16 weeks, though if people still had a low viral load after that time, they had the option of extending the ATI. It was discovered that one person in the CCR5-hetereozygous group in fact kept taking their ART and was excluded from the analysis.

There were no significant adverse events related to the study therapy. The single-dose electroporation technique was at least as effective as using the adenovirus vector: immediately after infusion, 25% of participants’ T-cells were CCR5 negative.

There were no cases of viral remission in this study: during the ATI, the HIV viral load in all subjects reappeared and the proportion of T-cells that were CCR5-negative slowly declined. There was a tendency for the CCR5-heterozygous subjects to retain a higher proportion of CCR5-negative inoculated cells. At four weeks, before the ATI, the proportion of T-cells that were modified was 4.6% and 4.1% in the two homozygous (both genes CCR5-positive) groups but 7.4% in the heterozygous group, and at week 24, four weeks after the ATI, the proportions were 2.2%, 2.6% and 4.6% respectively.

The cyclophosphamide appeared to make no difference to the time to viral rebound.

The heterozygous patients’ viral loads also reappeared more slowly. Whereas 50% of the homozygous patients already had a detectable viral load four weeks into their ATI, it took all 12 weeks before three out of five of the heterozygous patients did.

Three subjects had low viral loads at the end of the ATI and decided to prolong it. One homozygous patient had an undetectable viral load at the end of the ATI and delayed restarting until 12 weeks later, at a viral load of 10,000 copes/ml. Two of the heterozygous patients had viral loads in the 1000 copies/ml region and did not restart their ART till 20 and 32 weeks after their ATI respectively, at viral loads of 8000 copies/ml.

So while this experiment produced no patients with prolonged viral suppression, this is a demonstration of a safer, more repeatable and non-toxic way of creating a population of HIV-resistant T-cells that can be infused back into the body and which can to some extent delay HIV viral rebound, even when they only form less than 10% of the body’s complement of T-cells.

Asked about future steps, Dr Tebas said that instead of simply repeating the same techniques to engineer CCR5-negative cells, his team would investigate using ZFN technology to generate cells lacking the other HIV co-receptor, C4CR4,  and eventually to generate CRT T-cells – Chimeric Antigen Receptor cells, a type of artificially engineered T-cell already being used in anti-cancer therapy which will be able to seek out and destroy HIV infected T-cells.


Tebas P et al. Delayed viral rebound during ATI after infusion of CCR5 ZFN-treated CD4 T cells. Conference on Retroviruses and Opportunistic Infections, Seattle, abstract 106, 2019.

View the abstract on the conference website.

Watch the webcast of this presentation on the conference website.