Shutting the door to HIV? (part 2 of 2)

This is post two of a blog on HIV gene therapy; I’d highly recommend post one to help with understanding of what is to come, that being the discussion of a study in which CCR5 is deleted from cells of HIV infected individuals.

Over recent years ‘gene editing’ has started to take off, with new technologies being developed and made safe. One such approach uses artificial proteins known as zinc finger nucleases (ZFNs). These proteins are a combination of two naturally occurring proteins, a zinc finger protein and a nuclease (known as Fok1). Zinc fingers bind to specific stretches of DNA, while a nuclease is an enzyme capable of cutting through double stranded DNA. Combining the two produces a protein that can cut through DNA at specific locations.

A diagram demonstrating the principle of
ZFN cleavage of DNA

When breaks are made in both strands of DNA, a cell attempts to repair the damage – with one of three outcomes. The cell dies, the DNA is stitched back together and the original sequence is preserved, or the DNA is repaired with the random insertion or deletion of parts of the original base sequence. The third of these outcomes leads to the deletion of a gene. Using ZFNs to damage DNA at specific sites therefore allows for deletion of a specific gene – such as CCR5.

This leads me to the aforementioned study. In the work, published in the New England Journal of Medicine, HIV infected patients had blood taken, CD4 T-cells collected and then treated with a ZFN to specifically delete CCR5. Following the procedure, these cells were then infused back into the patient.

Twelve individuals received the ZFN treatment, in a study designed primarily to determine if this was safe. To that end they mostly achieved their aim. The majority of the patients in the study developed only mild reactions with 130 reported side effects such as, fever, chills, headaches etc. Out of these, only 32 of the side effects were linked directly to the modified cells, with the remaining number being attributed to the infusion process. Sadly one of the twelve did develop a severe adverse reaction and needed hospitalisation, but this was a response to the infusion, rather than the modified cells themselves. It would be nice to have none of these issues, but, as with any medical procedure, there is a likelihood of side effects, and the ones caused by infusion of the CCR5 deleted cells are largely acceptable.

So what impact did these cells have? As you may expect, the infusion of 10 billion or so cells caused an increase in the number of CD4 T-cells (the loss of CD4 T-cells is a hallmark feature of HIV infection). The mutant cells were detected at a reasonably high level, making up 13.9% of the circulating CD4 T-cells at any given time. Furthermore, the mutant cells were estimated to stay in an individual for close to 96 weeks. At this stage it’s clear that the infusion of CCR5 deleted cells is largely safe, and that these cells can survive in the blood for a fairly long period of time.

The main aim of this study was to assess the safety of CCR5 ZFN treatment, but there was also some investigation into the impact these CCR5 deleted cells had on HIV levels. All of the patients were on antiretroviral therapy at the start of the trial, and, as a result, had undetectable levels of HIV. However, six of the twelve patients were taken off their medication for twelve weeks, four weeks after the infusion of CCR5 deleted cells. Initially these patients showed virologic rebound, the phenomenon where HIV levels start to rise as soon as therapy is stopped. A peak in this was seen at six to eight weeks following cessation of therapy. However, at that point the levels began to decline – perhaps, optimistically, suggesting a protective effect from the CCR5 deleted cells. Moreover, as CD4 T-cell death occurred from the increased level of HIV, it was noted that the CCR5 deleted cells died at a slower rate than normal CD4 T-cells, further suggesting these cells may be protected from HIV infection.

At this stage no real conclusions can be drawn about the efficacy of infusing CCR5 deleted cells for treating HIV. This study achieved its aim to demonstrate safety of the procedure, but little can be learned about any protective effect at this stage. However, that is not to say this isn’t a promising avenue for HIV therapy in the near future.

An artistic representation of HIV showing the outer layer being
peeled away to show the inner core

A few aspects still need to be ironed out. For instance, of the approximately 10 billion cells infused to the patient, only 20% of these had the CCR5 deletion, meaning there are still a large number of cells for the virus to infect. Furthermore, the patients will continue to make their own immune cells from their own stem cells, which won’t have the CCR5 deletion, and will therefore, effectively, dilute the impact of the CCR5 deleted cells. Continual infusions may therefore be necessary for any protective effect. Alternatively, it may be possible to use a similar technique to engineer a CCR5 deletion into stem cells, allowing the patients to develop modified cells for the rest of their life.

Another issue may arise from the fact that HIV is a rapidly evolving virus, capable of becoming resistant to any treatment given in isolation. I would speculate that if a patient, infected with HIV were to be taken off all medication and given CCR5 deleted cells, then HIV viruses that bind CXCR4 would become more prevalent, or perhaps some other work around would evolve. What impact this may have is unknown at this stage.

No doubt a large scale, phase 2 clinical trial is, soon to be, or already underway to assess the protective effects of the CCR5 deletion against HIV over a longer time period of time, with a larger number of patients. It will be interesting to see how much clinical impact this procedure can truly have. And what ways the virus may find to escape the intervention.

Shutting the door to HIV? (part 1 of 2)

Earlier this month a study was published that suggested a potential new approach to treat HIV infection using genetic modification of the T-cells HIV infects. This study rightly generated media interest, so I thought I’d give my take on it and outline some of the details. I’ve divided the post into two parts, allowing for an ample amount of background to aid understand of the details of the study, without scaring you off with one long post.

To begin with, a bit of cell biology. A protective layer of lipid molecules, in a double sheet, surrounds our cells. This ‘lipid bilayer’ provides separation between the inside and outside of the cell. However, cells needs to interact with the environment and transport molecules across the lipid bilayer. One of the major mechanisms to have evolved are cell surface receptor proteins. These proteins reside in the lipid bilayer, exposed to the environment, and can bind molecules, in some cases these are then transported into the cell. These proteins are essential for the normal functioning of our cells.

A schematic of a lipid bilayer with inserted proteins
spanning it (transmembrane proteins)

Like other viruses, HIV must enter cells to replicate. Without wanting to overly anthropomorphise the situation, viruses are devious in the ways they do this. In the case of HIV, binding to two cell surface receptors drives a shape change in a protein on the virus that causes it to fuse with the lipid membrane of the cell. This fusion event allows entry of the viral genetic material, which can then undergo replication.

CD4 is the essential receptor for HIV; it is the first receptor the virus interacts with. CD4 is a protein expressed by cells of the immune system, such as T-cells and macrophage. Once bound to CD4, the virus must bind to one of two co-receptors, either CCR5 or CXCR4. When this second binding event has occurred, the shape change will happen, and the virus will fuse with the cell and enter.

HIV fusion

The vast majority of viruses that are transmitted between individuals, and many of the viruses within an individual are CCR5 tropic (they bind CCR5 and not CXCR4). You could therefore speculate that without CCR5, HIV transmission would be blocked, and infection levels reduced. Without CCR5 on cells, could we shut the door to HIV infection? However, an important question is whether this would be safe? Can people survive without CCR5?

Interestingly, around 4-16% of people with European descent have a mutation in at least one copy of the CCR5 gene (CCR5 – italics denote a gene) known as CCR5 delta 32 (there is deletion of 32 base pairs from the gene). People with this mutation in one gene copy, 'heterozygotes', express roughly half the level of the protein compared to a person with no mutation. It is also possible to have the delta 32 mutation in both copies of the gene, 'homozygotes', leading to no CCR5 protein being present on any cells – and these people are perfectly healthy. What’s more, people carrying CCR5 delta 32 mutations have resistance to HIV infection, and slower progression to AIDS if they are infected, particularly the homozygotes.

The hypothesis that disruption of CCR5 could be protective was further strengthened in 2008 with the ‘Berlin patient.’ This individual, later disclosed as being named Timothy Rae Brown, has been functionally cured of HIV. Brown was diagnosed with HIV in 1995, and was subsequently diagnosed with a leukaemia in 2006. The leukaemia could be treated with a bone marrow transplant. Brown’s doctor, Gero Hütter, decided to acquire the bone marrow from a donor homozygous for CCR5 delta 32. Stem cells of the immune system reside in bone marrow; so receiving bone marrow from a homozygous CCR5 delta 32 individual meant Brown developed an immune system populated by cells containing the mutation. In 2009, Brown had been off his antiretroviral therapy, used to treat HIV, for over a year and had had no detectable virus. He remains, to this day, free from antiretroviral therapy and still has no detectable HIV.

As amazing as the story of the Berlin patient is, bone marrow transplants are not a realistic option to treat HIV infection because of cost and the risks associated with the procedure. However, what if there were other, less drastic, ways of introducing mutations to CCR5?

This is where I’ll leave the first part of this post. Tomorrow I’ll post part two looking in detail at a study assessing the safety of genetically modifying cells to cause deletion of CCR5, and to see the impact this has on HIV infection. So come back tomorrow for more…

Pithovirus - More than just a large storage container

I have a fascination with giant viruses; they have made two previous appearances on my blog (here/here and here). The reason for this fascination is two-fold; firstly, their discovery shattered the notion of viruses as tiny, sub-microscope, particles and secondly, because they have resulted in some intriguing ideas surrounding the beginning of life itself and even the suggestion of a fourth domain of life (see the two previous posts for more details). It has been over 10 years since the first of these giant viruses, Mimivirus, was characterised. Following that discovery, multiple related viruses were identified, and the Megaviridae family was defined. It was thought that these viruses were the only family of giant viruses, one (literally) huge exception amongst a plethora of viral families. That was until the discovery of Pandoravirus, a virus that was even larger than any members of the Megaviridae, and, moreover, completely different. This opened the door for there to be more families of giant viruses, the Megaviridae were no longer an exception. A third viral family has now entered the furor, sitting somewhere between the two previously defined families - this is Pithovirus.

The name derives from the Greek pithos meaning ‘large storage container,’ and was chosen for the fact that a pithos was given to Pandora (Pandora's box) - hinting towards the link with Pandoravirus. The two viruses look very similar as you can see in the images below. Additionally, the naming, either deliberately or fortuitously, hints at another feature of Pithovirus - that being its size. It is even larger than Pandoravirus (the previous giant virus record holder) measuring 1.5 micrometers in length and 0.5 micrometers wide - making it a ‘large storage container’ indeed (as viruses go anyway). To put that size in context, influenza virus is roughly spherical, with diameter of approximately 0.1 micrometers.

Pandoravirus (false colouring for display)

Pithovirus (false colouring for display)

What's even more intriguing about the size of Pithovirus is the fact the genome is comparatively tiny. Pandoravirus measures at 1 x 0.5 micrometers, and weights in with a genome of 2.8 million base pairs of double stranded DNA (the same genetic building blocks we use). However, the physically larger Pithovirus has a genome of only 610,033 base pairs - about 4.5 times smaller than Pandoravirus. Additionally, while the two viruses may look similar from the outside, their genomes are completely different. DNA is made up of bases, of which there are 4: A, T, C and G. The genome of Pithovirus is 64% AT, whereas Pandoravirus is 61% GC. Furthermore, there are only 5 proteins made by Pithovirus that have any similarity those of Pandoravirus.

It seems strange that Pithovirus has such a large shell to encase, comparatively, such little genetic material. The virus Phaeocystis globosa has a similar sized genome, but this is encased in an icosahedron of only 0.15 micrometers in diameter.

One possible explanation for the discrepancy between the viral particle size and genome size could be due to gene loss - an idea suggesting that viruses originate from independent cellular organisms that lose genes and become dependent on a host. Perhaps Pithovirus has lost genes and become parasitic, but has not evolved into a small particle. (See the Pandoravirus blog for more details about the idea of gene loss).

An alternative explanation for the size of Pithovirus could be that this feature is essential for its lifecycle. Pithovirus is a virus that infects amoeba, and indeed, it's ability to infect Acanthamoeba castellani was how it was discovered. This is something in common between all the giant viruses - they all prey on amoeba. Amoeba are single celled organisms that obtain nutrients (eat) through a process known as phagocytosis - the same process our macrophages use to engulf foreign organisms and protect us from infection. Could it be that these viruses need to be giant to ensure that amoeba confuse them for nutrients and phagocytose them? Without being phagocytosed, the virus would not be able to enter the amoeba and could not replicate. Viruses are incredibly adept at subverting essential mechanisms of a cell to their own end, so perhaps the sheer size of these viruses is another example of this. It will be interesting to see, as more giant viruses are discovered, whether any can infect organisms that do not rely so heavily on phagocytosis. 

Not only is the size of Pithovirus fascinating, but also how it was discovered. The team, working from the labs of Jean-Michel Claverie and Chantal Abergel, obtained samples of Siberian permafrost dating back approximately 30,000 years. Using these permafrost samples the teams then inoculated cultures of Acanthamoeba castellani and found that Pithovirus was able to grow from these cultures. What is truly remarkable about this is that Pithovirus had been frozen in Siberian permafrost for over 30,000 years and could be thawed out and become infectious. This makes it the oldest infectious organism ever discovered.

The discovery of Pithovirus in 30,000-year-old permafrost does beg the question about what else might be lurking in sediments hidden away in the Siberian ice. Climate change in the Russian Arctic is more evident than many other parts of the world, having seen a 3°C temperature increase in the last 100 years, compared to a global average increase of 0. 7°C. As the ice thaws, it may be possible that other ancient infectious organisms could be released. While this study does not say that this will happen for sure, it makes it clear that surveying permafrost regions may be an important scientific expedition, not just to detect anything dangerous, but also to potentially uncover new and exciting organisms.

The discovery of Pithovirus is fascinating as it adds yet another layer of complexity to our understanding of the giant viruses. It seems to sit somewhere in between the two previously describe families, looking like Pandoravirus, but being genetically more closely related to members of the Megaviridae. It was a pithos that Pandora received from the Gods containing all the worlds’ evils. While Pithovirus is not going to bring any evils to anything other than amoeba, the fact that a virus can be preserved for that long in Siberian permafrost does come with a warning that more exploration may be important. And not only important, but also exciting, who knows what we may find!