Protection from suppression

I know very little about the demographic of the readers of this blog, however, I would fancy my chances betting that the majority of readers haven’t recently received an influenza vaccination, I certainly haven’t. The main group of people that receive the vaccine (here in the UK anyway) are those considered most “at risk” – these predominantly being the very young and old or those with underlying health problems.

Illustration of an influenza viral particle and
an enlargement of the HA spike

Vaccination largely works by stimulating the production of antibodies that target a specific part of a pathogen. In the case of influenza, the main target for these antibodies is the hemagglutinin (HA) spike, which is exposed on the surface of viral particles. As you can see in the picture, the spike is made up of a ‘head’ and a ‘stalk.’ The vast majority of antibodies produced are against the head. However, this has issues because the head can easily mutate and become unrecognizable to the antibodies we produce. As such, a new influenza vaccine must be given every year so to generate novel antibodies against the newly mutated virus. Annual vaccination has issues in the fact that the vaccine must be produced in advance of the “’flu season,” necessitating a degree of guesswork about the viruses that will be circulating in the coming winter months. Furthermore, should a completely new virus emerge, there will be no protection provided by the current vaccine – as was the case in 2009 with the Swine Flu pandemic (a H1N1 virus), and as is feared for Bird Flu viruses (either H5N1 or H7N9).

The best solution to the issues of the current vaccine is to develop a ‘universal’ vaccine. In an ideal world, this would be a single shot vaccine that would provide life-long protection against all influenza viruses. If this could be achieved, there would be no need to give new vaccine doses every year and there would be substantially less fear about pandemic spread of a new virus. A universal vaccine would need to produce antibodies against part of the virus that is conserved between all influenza stains (e.g. is the same in a H1N1 virus and a H7N9 and a H16N10 and so on) and, by extension, does not easily mutate. A vaccine fitting these criteria will produce a ‘cross-protective’ response. Fortunately, the HA stalk has these properties and could, in principle, be used as a universal vaccine that would provide protection against all influenza viruses. The only problem is no vaccines have been able to produce antibodies targeting the stalk.

This brings me onto the main topic for this blog; a paper recently published in the journal Nature Immunology from the labs of Paul Thomas and Maureen McGargill. I felt the need to blog about this paper based on some very interesting findings about the immune response to influenza. In essence, their work found that treatment with an immunosuppressive drug, known as rapamycin, is able to cause a cross-protective response, thus enhancing the prospect of generating a universal vaccine.

In their study, the vaccination was of mice using a weakened H3N2 virus and then subsequently infecting with a H5N1 virus to test the efficacy of the vaccine. The team found that if the mice were treated with rapamycin prior to the vaccine, they would be protected against the H5N1 virus. In contrast, the mice not given rapamycin mostly died within 2 weeks of H5N1 infection. Importantly, they also showed that treatment with rapamycin and then infection with H5N1 (without the H3N2 vaccine) did not provide any protection. Therefore, something about the primary infection with H3N2 virus was being modulated to provide better protection against H5N1.

This led the team down a path to try and find the culprit for this protection, which turned out to be B cells (the immune cells responsible for producing antibodies). Without B cells, and therefore antibodies, mice had no protection against H5N1 following the H3N2 vaccination. Additionally, it was seen that, following rapamycin treatment, the antibodies produced were targeting the HA stalk, instead of the head. Therefore, the question to ask is: how is rapamycin altering the antibody response?

Antibodies fall into 5 classes known as Ig (immunoglobulin) M, G, A, D and E. When the immune response is first triggered, for example by the H3N2 virus, the vast majority of antibodies produced are of the IgM class. Over time a process of ‘class switching’ occurs that produces an improved immune response. Against a viral vaccine, most class switching from IgM produces IgG antibodies that have a higher affinity for the virus and therefore improve the protection. It was found that rapamycin treatment inhibited class switching, causing an excess of IgM antibodies in the rapamycin treated, vaccinated animals.

The reason I wanted to blog about this study is that it raises some interesting points about evolution. Class switching is important, if we didn’t have it, our immune system would be nowhere near as effective at protecting us from pathogenic infection. It wasn’t looked at, but I would speculate that any rapamycin treated mice that were vaccinated with the weakened H3N2 and then infected with a H3N2 virus would have a slightly weaker immune response against the re-infection compared to animals that could class switch. However, blocking class switching has the advantage of producing a cross protective response. Since we have class switching, it appears that animals have evolved to produce the strongest, and most specific immune response they can, in the most specific manner possible – all the eggs are in one basket as it were. Evolution has no foresight so only selects for the animal that has the best protection. Furthermore, it would seem to suggest that in evolutionary history there may have been less circulating influenza viruses as the production of a cross protective response was not actively selected for. The final point to make is that this may explain why the stalk region of the HA spike is highly conserved between influenza viruses, compared to the head. If the best immune response produced is by targeting the head region of HA spikes, then it is advantageous for the virus to mutate this. If our immune system rarely targets the stalk, there is no evolutionary pressure for this to rapidly mutate.

In the vast majority of cases, producing a very strong immune response is essential to our survival from infection. However, this study points towards a potential new avenue to boost our search for a universal influenza vaccine, by counter-intuitively blocking the evolutionarily advantageous process of class switching.

Pandora's box is viral

Anyone who reads my blog regularly will know my fondness of viruses (they feature in most posts). I am particularly curious about giant viruses and have previously posted about these (link one and two). The giant virus field has recently grown larger (pun completely intended) with the addition of a new family of even larger viruses, so I thought it was about time to update the story.

Pandoravirus

The recently discovered viruses are known as Pandoraviruses and are not just causing excitement because of their size. But let's start with their size. A conventional microscope cannot see the majority of viruses, unlike bacteria and eukaryotic cells (such as our own). Their size is usually in a range of 50-150 nanometers (influenza virus for example is about 100 nanometers in diameter). A nanometer is millionth of a millimeter - the average pinhead has a diameter of 2 millimeters, meaning the average virus is around 20,000 times small than a pinhead. Another comparison would be to bacteria, which are usually around 1000 nanometers in length. Pandoraviruses are not like most viruses; they stands at a whopping 1000 nanometers long and about 500 nanometers across – dwarfing nearly all other viruses and even some bacteria. They don't even look like viruses! It's quite possible these viruses were discovered 13 years ago, but were not appreciated for what they truly are.

Know your sizes

 As if the sheer physical size wasn't enough, let's consider the genome size. We humans have a genome of around 3 billion bases (a base being the individual unit of DNA), which encode between 20,000 and 30,000 genes. Bringing things down to the virus scale, HIV-1 has a genome made of around 10,000 bases, which code for 9 genes. Influenza is slightly larger, having a genome of around 14,000 bases, coding for 11 genes. Viruses are traditionally known for being small and not carrying much luggage in their genetic material. In stark contrast, Pandoraviruses have up to a staggering 2.5 million bases, coding a total of 2556 genes. This is more genetic material than a lot of bacteria and even some parasitic eukaryotic organisms.

Comparison of genome sizes for different domains of life (and viruses)

Size matters, but that is by no means the most interesting thing about Pandoraviruses. All life on Earth is classified into three domains on the so-called tree of life: eukaryotes, bacteria and archaea. When the genes and proteins of all these different domains are compared there are strong similarities. Eukaryotes tend to have the most complexity, but at the core, certain things are the same. For instance, imagine a protein that has a structure of AABCDD in a bacterium. This may be the simplest form of that protein. We humans may have a very similar core structure but have, through evolution, made it more complex – for example, AAA*BCD*D*EEFG (where the * is denoting small changes to that unit). While this is different it still carries strong similarities to the simpler, bacterial protein. These similarities point towards the fact that all life on Earth may have originated from a single point – the seed for the tree of life – which has then branched out through evolution to give all the life we see around us.

Basic diagram of the three domains of life

Viruses are not considered living; so do not fit onto the tree of life. However, they do adhere to the rule of thumb of having strong similarities to other organisms. While not living, they are still similar to the living. Again, that is the story for most viruses, but not the Pandoraviruses. Only 7% of the Pandoravirus genome has similarity to our existing database of proteins and genes. This means 93% of the proteins Pandoviruses produce have no similarity to anything we have knowledge about…

This complete lack of similarity to any other form of life raises something potentially very fundamental about our view of the world. There is a view that these giant viruses evolved in a reductionist manner. Evolution is often thought of in a unidirectional manner, gaining increased complexity. However, this is not a rule of evolution, the rule of evolution is the selection of advantageous traits. If becoming less complex provides an advantage then this will be selected for. The hypothesis surrounding the giant viruses is that they were once free-living cells (similar to a bacterium for example) that through evolution lost their self-sufficiency, instead becoming dependent on a host to reproduce - a trait of a virus. If this were the case then giant viruses would need to be related to other forms of life, since would have originally been a life form unto themselves. However, as I've said, there is only 7% similarity between Pandoraviruses and all other life forms. This means that the ancestors of Pandoraviruses were probably very different to the ancestors of bacteria, archaea and eukaryotes. Therefore, where do they fit in our view of evolution? One resolution of this issue is that maybe there aren't (or at least weren't) only three domains of life. Pandoraviruses and the other giant viruses could represent the modern day descendants of a forth domain of life. This is merely an idea with no real concrete evidence, but it seems exciting to me that as we learn more about these giant viruses, and as we discover more (which I'm sure we will), our view of life on Earth may need to change. As has often been the case in biological science, viruses could be paving the way to a huge shift in our understanding of the world around us. 

A perfect iFITm to fight viruses

There are an estimated 7 billion humans worldwide. That's 7,000,000,000, a huge number. However, 7 billion pales in comparison to the estimated 10 to the power 31 viruses living alongside us - that's 10,000,000,000,000,000,000,000,000,000,000. Put another way, for every predicted star in the universe there are 10 million viruses on Earth. Closer to home, within each of us there are approximately 4 trillion viruses; yet for the majority of our lives we will be healthy and free of viral disease. There are many reasons for this, not least because most viruses have no interest in humans. But another major factor is a set of cells and molecules to which we are deeply indebted: these are the components that make up our immune system.

The immune system can be broadly divided into two interlinked halves. There are the front line, rapid responders comprising the innate immune system; cells and molecules that are evolutionarily "designed" to react rapidly and broadly, controlling invasion from any foreign organism. The second half is the adaptive immune system. This has the big guns, taking longer to mobilize but with the power to fully clear invading organisms following the initial suppression from the innate system. Together these two halves co-ordinate a response that effectively removes almost all infectious agents we encounter. Every once in a while we will get briefly sick as the system kicks into full force, but largely, we will never notice the silent protection it provides. The halves are inseparable. However, without the rapid response of our innate system many viruses and other pathogens would be able to gain a foothold and potentially overwhelm us. Furthermore, the adaptive immune system cannot be triggered unless the innate system warns it of an invasion, making the innate immune system paramount for our survival.

Possibly the most important players in the innate response to viral infection are a group of proteins known as interferons (IFNs). Following an infection, IFNs trigger the expression of 200-300 genes, producing effector proteins that function as part of the innate immune response to block viral infection. It is known that these IFN stimulated genes protect us, however, very little is known about the function of individual proteins produced from these genes. In 2009 a major new component in the IFN response was discovered that is now known as InterFeron-Induced TransMembrane protein 3 (IFITM3). This protein was found through its ability to protect cells from influenza virus infection. Influenza virus, or flu, annually causes 3-5 million cases of clinical disease worldwide, and has the major potential for pandemic spread. It was subsequently found that IFITM3 is present in the cells of our lungs (the site of influenza infection) and that these cells can be stimulated to express even more of this protective protein following infection.

 

Influenza virus particles

Most importantly for the story of IFITM3, it was found not only to protect cells in a laboratory, but also to protect us on a regular basis. Usually flu is a minor illness, you simply get over it after a couple of weeks. However, certain people can be hospitalized, or worse. In a study of patients hospitalized by influenza, it has been found that a mutation in IFITM3 was over-represented. To put it another way, it seems that people who carry a mutation in IFITM3 are at an increased risk of severe flu infection.

What makes IFITM3, and its close relatives IFITM1 and IFITM2, even more exciting is that they don't just protect against influenza. At the time of writing, the list of viruses the IFITMs protect against is in double figures and includes some of the most notorious known to infect humans. Dengue virus, a mosquito borne virus that threatens around 3 billion people, is sensitive to IFITMs. Similarly, Ebola virus that can kill up to 90% of the people it infects is restricted by the IFITMs. HIV may even be kept in check by the IFITM proteins. This is to name but three. Obviously these viruses still cause infections and still kill on a daily basis. However, without protection provided by the IFITMs it is possible these viruses could be even greater killers.

Currently, we still do not completely understand how the IFITM proteins work. If this is fully elucidated we may be able to produce drugs that mimic their protective properties. Better yet, we may be able to regulate expression of the IFITMs or improve on their function and provide even greater levels of protection. Imagine if we could make a single drug that protected against infection by the four viruses already mentioned and a whole host of others! This may be slightly blue-sky thinking, but as someone at the start of a PhD in the field, that sky looks pretty bright and appealing to me. 


(NB. This article was entered into the Max Perutz Science Writing Competition run by the MRC by myself and is my own work in both cases)

Adding some culture (and history) to the blog

As someone starting a career in a biological field of science I find it hard to imagine a world in which it wasn't possible to do experiments using human cells, it's pretty much all I do. However, until the 1950s the use of human cells in a lab was only done in a few places that had the expertise. Following the establishment of techniques to grow cells in a lab, and the discovery of immortal cell lines that grow forever, biological science exploded! Almost all of the major breakthroughs of the last 60 years will have at some point used cells in a lab. So I though I'd make a post talking about the history of cell culture (as it is known).

A cell culture hood used to grow cells in sterile environment

To start with an important question - why do we want to work on cells? A cell is the building block of life; humans are made of close to 10 trillion of them, each containing the genetic code that turns to us into who and what we are. Since cells underpin all of life they are of the upmost interest to sciences studying any biological question. However, studying cells within a living being is pretty difficult. Human experimentation is largely frowned upon, so we look to animals and more simple organisms to guide or understanding. Even then you're looking at a whole system made up of hundreds of different cell types all interacting and maintaining life. The ability to look at a single type of cell provides much more useful information - if you were trying find when a specific person was born, you wouldn't want to have to deal with a whole city of people. 

This desire to look at single cells outside of a living being was the spark that set scientists on the road to develop cell culture. It all began in 1885 when Wilhelm Roux managed to keep cells from an embryonic chicken alive in a laboratory. They didn't last long, but it was a start. Not for want of trying, but it wasn't until 1907 that the next major breakthrough was made by Ross Harrison who managed to actually grow cells in his lab, rather than just keep them alive. Similarly to Roux, Harrison did not use human cells, instead opting for nerve cells from a frog, and his cells didn't last long.

Another big gap ensued. It wasn't until a period through the 1930s-1950s that cell culture, and particularly that of human cells, really took off and set the foundations for almost all biological science to this day. To a large extent this work was dictated by the hunt for a polio vaccine. To produce a vaccine it is necessary to produce virus particles, which can then be injected into a recipient to produce a protective immune response. There was however a hurdle for this at the time. Viruses need cells in which to grow, and a vaccine needs lots of virus particles. In the early 1930s, the best way to grow virus was to infect an animal and collect samples. This was indeed done under the guise of vaccine development by infecting monkeys and collecting extracts from the nervous system. However, the collected viral particles were contaminated with monkey nervous system extract, which was found to cause paralysis when injected into humans, the very syndrome a polio vaccine should prevent. This approach was rapidly binned as you can imagine.

Following the failure of this monkey grown virus, attention shifted to growing virus in the safer and more controlled environment of a laboratory, using human cells. This has many advantages of which safety is a huge one. But additionally, there is a huge economic benefit - it is much cheaper to look after cells in a lab than a whole collection of animals. This desire led to work in 1936 by Albert Sabin and Peter Olitsky, at the Rockefeller Institute in America, who grew poliovirus in cultures of human brain tissue. While this was an achievement, they were concerned that using brain tissue might have similar issues to those seen with monkeys. As such, they attempted to recapitulate their results in other cell extracts - but to no avail.

Cartoon of poliovirus

It wasn't for another 13 years that the aim of growing poliovirus using human cells in a lab was realized. This advancement was a complete accident by John Enders, Thomas Weller and Franklin Robbins. At the time, these eventual Nobel Laureates were attempting to grow varicella virus (chicken pox). They had managed to grow human skin and muscle cells in a lab and infected these cells with varicella. As a control virus, used because they thought it wouldn't grow, so acted as a point of comparison, they used poliovirus. In a huge stroke of luck, or mis-fortune depending how you see it, their original experiment failed. Varicella failed to grow, however, poliovirus did! Following this, these men moved on to grow the virus in multiple different extracts of human cells and eventually managed to get a high level of production of the virus (earning them their Nobel Prize). This work paved the way for Jonas Salk to produce the first polio vaccine.

The science of growing human cells in a lab had advanced a long way by the start of the 1950s. From a point of relying entirely on live animals, it was now possible to grow cells extracted from humans and use these to produce the viruses needed for vaccines. However, these cells still had issues. The way these cells were produced was largely from biopsies of human tissues. In a biopsy, a small chunk of tissue is taken from the body, such as the skin. These chunks of human material were then broken up into individual cells and grown in a lab. The issue comes from the fact that biopsies take all the cells that reside in a certain area, giving a very mixed population of cells. This limits the reproducibility of experiments since different tissue samples will be made up of highly variable cell types. Reproducibility is a bedrock of the scientific enterprise so this was hardly ideal. Additionally, the cells only lasted for a short amount of time when grown in a lab, meaning there was a need for lots of biopsies.

HeLa cells

In 1951 (and over the following years) these issues with human cell culture were overcome when the first ever immortal cell line was produce - biological science hasn't looked back since. 1951 saw the birth of the HeLa cell line which is still the most commonly used cell line in the world with an estimated 60,000 scientific articles published using these cells as of 2009. HeLa is taken from the name Henrietta Lacks from whom these cells were extracted. Lacks died in 1951 from an aggressive adenocarcinoma of the cervix, from which the cells were taken in the form of a biopsy. Lacks’ physician gave the extracted cancerous cells to George Otto Gey, who set up the cells as any other biopsied tissue to grow in a lab. To his surprise the cells survived with ease, and continued to grow indefinitely - they are still growing. These cells were taken like any other biopsy (as discussed above), but what made them truly special was that they came from cancer instead of healthy tissue. Cancers are a clonal diseases, meaning that all cancerous cells are (essentially) exactly the same. This clonality gave the all-important reproducibility so craved by scientists since everyone could work on cells that were exactly the same. What’s more, these cancer cells had a mutation that made them immortal (given the right treatment), meaning they can grow indefinitely outside of a body.

Sadly, HeLa cells are shrouded in controversy. Lacks' physician did not ask for her, or her family's permission to donate the cells to Gey. By the time they were made aware, the cells had been patented and commercialized and had taken over science. Companies had made millions of dollars selling the cells and the Lacks family didn't see a dime. In America this is still allowed, there is no need for a physician to obtain permission to use cells extracted from a patient. However, in the UK there is a need for ethical approval and patient consent.

While there are certainly some injustices surrounding the development of HeLa cells, the contribution of Henrietta Lacks to science is unparalleled. To his credit, Gey freely gave away the cells to anyone who wanted them for the "advancement of science". Tying things together – shortly after their development the cells found their way to Jonas Salk who used them to test the safety of his polio vaccine.

The cells have since been used for the testing and production of many other vaccines; they have also been used for the production of drugs to treat cancer and HIV (and many other viruses). They have gone to space and have been subject to Chernobyl-esque levels of radiation. Since we can't test most things on a human straight away, having humans cells growing in a lab that you can throw everything but the kitchen sink at (there’s an issue with proportions there) gives a fantastic way to test out how safe (or dangerous) something is to humans.

Plates in which cells can be grown

The ability to grow and study cells in a lab has contributed an incredible amount to science. Being able to grow cells extracted from humans lead to early discoveries in virology that paved the way for numerous vaccines. With the discovery of immortalized cells, such as HeLa, the whole thing became even easier and more uniform, giving the reproducibility scientists crave. The story of HeLa is one of controversy as well as being a world-changing discovery. I've attempted to give a taste of the HeLa history here, but for anyone wanting more, I highly recommend "The Immortal Life of Henrietta Lacks" by Rebecca Skloot. This is not to advertise the book (and there is no conflicting interest - in the desire for full disclosure), but reading that gave me the impetus to write this blog and I feel credit should be given where it is due. 

With all this written, it's time I go back and look after my cells.