Build your own vaccine

 RNA technology has reshaped the vaccine landscape. The biggest advantage is the flexibility. Every single virus, bacteria, fungus or parasite that has ever infected a human uses the same code set to make its proteins. It’s basically universal science Lego. This means we can build vaccines against any pathogen using the same tool kit.

In theory.

The first generation of RNA vaccines were highly effective during the pandemic, they significantly reduced the burden of disease and helped protect many people from hospitalisation and death. But more can be done to improve them for future pandemics or more routine use. One area that requires attention is how the RNA is delivered to cells.

RNA is a large, charged and fragile molecule. If injected into the body on its own, it gets chewed up; this is a natural defence system from the body to protect it against viruses, bacteria, fungi and parasites (which as mentioned above, all use RNA too, and would like nothing more than our cells to make their proteins). We therefore need to package it up to deliver the vaccine payload.

As part of a collaboration with Professor Cameron Alexander (University of Nottingham) and a Cambridge biotech company called Aqdot, we explored a new approach to package RNA. This was recently published in the journal Advanced Materials. The licensed RNA vaccines (from Pfizer and Moderna) use a delivery technology called LNPs. These are Lipid NanoParticles – they use fat particles to encapsulate the RNA, a bit like salad dressing (but not really). There is an alternative approach, that exploits the charge of the RNA molecule. It is long and negatively charged, this means it can then be combined with long positively charged molecules. However, long positive molecules can be toxic to cells – because they bind other stuff, disrupting normal function. The breakthrough in our published study: ‘Modular Supramolecular Polycations Enable Efficient Delivery of Diverse RNA Therapeutics and Vaccines’ was a novel way of packaging RNA, developed by the chemistry team at Nottingham.

Instead of using long molecules, they used much shorter ones, but used a linker to join it all together. The linker was developed by Aqdot – it is from a family of molecules named cucurbiturils (so called because they vaguely look like cucumbers). The cucurbituril molecules are barrel shaped and the hole in the middle is able to accommodate other polymers. This can then link everything together in a web – including the RNA. Using the new platform, we were able to show that you can deliver RNA vaccines and protect against infection with influenza.

There are three major challenges with RNA vaccines – the reactogenicity (side effects after immunisation), the longevity (how long the response lasts) and the stability (the need to keep them in -80). Much of this relates to how the RNA is packaged, the ability to deliver RNA with different formulations opens up opportunities for the further development of the RNA vaccine platform.

24 hours later: using systems vaccinology to understand responses to self-amplifying RNA

 Vaccination works by training the immune system to recognise parts of infectious micro-organisms, so that when we get infected with them we can better fight them off. This builds upon the immune system’s ability to remember what it has seen before. Training this memory takes some time from the initial encounter with the vaccine. Importantly, the events immediately after vaccination can shape the quality and quantity of the response.

These early responses to immunisation have been explored across a range of different vaccines. In our recent study ‘Systems vaccinology analysis of saRNA immunization identifies an acute innate immune signature correlated with adaptive immunity’ we measured the immune response at 24 hours to an RNA vaccine to explore how this might predict responses later on.

The vaccination part of the study took place as part of early trials of a self-amplifying RNA vaccine during the COVID-19 pandemic. This vaccine approach has potential advantages over the mRNA vaccines because the ability to amplify itself means the dose use can be much smaller. The study was performed towards the end of 2020 when the licensed vaccines were beginning to be offered widely to people. This reduced the number of people eligible for the study – because we wanted to evaluate responses in people who had not been vaccinated before. But we were able to enrol a small number of volunteers and undertook the study.

We used an approach called systems vaccinology. Which is a sciencey way of saying we measured lots of stuff and then looked for associations in the data. Blood was collected 24 hours after the initial injection. And three categories of immune markers were measured: RNA, protein and cells. The RNA gives a global picture of everything that has changed, the proteins focuses in on the way that immune cells communicate with one another and the cells gives a snapshot of what immune cells are moving from one place to another. These measurements because they are taken very soon after immunisation tell us about the short-term reaction to the vaccination, but the important thing is then to link them to the outcomes of vaccination. To do this we took a second blood sample several weeks later to measure whether the vaccine had indeed trained a memory response that could help prevent future infections.

We observed that immunisation led to significant changes in immune signalling in the blood. When the changes in RNA were profiled, there was a significant increase in genes linked to the immune response. Many of these fell in a family called the type I interferon pathway. This was not entirely surprising, RNA vaccines are made of RNA (#spoiler), so are many viruses; the type I interferon pathway is a programmed package of genes that fight viruses. We also saw an increase in genes that encode signalling molecules – particularly those that instruct immune cells to move from one place to another. These are called chemokines.

Having looked at the broad RNA picture, we focused down on proteins (as a quick reminder, RNA encodes proteins, proteins do the functional stuff). We saw similar increases in signalling molecules at the protein level in the blood, with increases in a chemokine called CCL2. One of the important functions of the immune system is to direct white blood cells (immune cells) to where the infection is occurring. CCL2 encourages the movement of a family of cells called monocytes, which have the ability to carry vaccine from one place to another and then engage with the cells that form the immune memory (called lymphocytes). Matching the increase in CCL2, we also saw an increase in the cells they recruit (monocytes) in the blood.

This demonstrated that the vaccine was somehow engaging the immune response, but did it mean anything with regards the training response? Well of course it did – otherwise I wouldn’t be writing about it. When we compared the magnitude of the CCL2 response in the blood with the magnitude of the antibodies targeting SARS-CoV-2 spike protein (the vaccine payload). People who had more CCL2 had more antibody.

So why is this important? It allows us to make predictions of how well a vaccine will work as soon as 24 hours after immunisation. It also enables us to design vaccines – if we can learn how to increase the induction of CCL2, we might improve the strength of the response. Of course this work was a massive team effort – and we owe a great debt to the volunteers, who during a time of considerable uncertainty undertook a study to help move us closer to having a working COVID vaccine.

All models are wrong, some are useful

 Cross referencing different systems can compensate for issues in individual systems and provide novel insight.

The saying about the limitations of models is attributed to a British Statistician, George Box. It speaks to how we use models to understand the world around us. This reflects is a deeper philosophical discussion as to what extent we can ever reach ‘truth’. But for now the point is that the models and observations we make in medicine and biology are often flawed. And we need to be aware of these flaws in order to better utilise the models.

In our recently published paper Comparative cross-species transcriptomics during RSV infection identifies targets to treat RSV disease we combined three different approaches to understand infection with Respiratory Syncytial Virus (RSV), a significant cause of illness in babies less than six months old.

Although natural RSV infection in children is the disease of interest, it is challenging to study directly in babies. It is very difficult to collect samples and it is often unknown when the child was first infected. An alternative is to use human infection challenge studies – where volunteers are deliberately infected with RSV. This has the advantage of being in the same species (the human) but the studies use young, healthy adults not babies. There is an additional challenge that all adults in these studies will have previously been infected with RSV at some time in their lives (probably several times) which will affect the immune response to any subsequent infection. The volunteers typically experience mild-to-moderate disease and so don’t fully recapitulate the disease seen in babies. Another alternative is to use mouse models. As well as the possibility to perform biological repeats in genetically identical individuals, following infection of a mouse, you can access all tissues and you can manipulate the response experimentally. However, there are interspecies differences in physiology, behaviour, viral tropism and genetics which can limit interpretation. All experimental approaches are ethically assessed, but there is a sliding scale – more can be done in adults than babies, more in mice than humans. And so by combining these different approaches we can compensate for limitations inherent to each.

In the published study, we compared the immune response in the blood, the lungs and the nose. As with different models, sampling different sites compensates for limitations of sampling an individual site. Blood is easily accessible and it is possible to collect large volumes of material, multiple times. But for a respiratory virus like RSV, it isn’t the actual site of infection (even though the lungs are highly perfused with blood). Blood can reflect cells moving into or out of the lungs, but not the complete picture. The lungs, as the site of infection tell us what is happening where the virus is and therefore can give us much more information. However, they are much harder to sample – it takes a medical procedure called a bronchoscopy to collect the tissue. Repeat sampling over time is not possible and these kind of samples cannot be collected from babies. The nose represents a good compromise. It is easily accessible and is the entry site for infection. Newer sampling methods have enabled the collection of good quality material from the nose without causing discomfort.

Having collected material from infected individuals, we then measured changes in gene expression. Cells, when they are infected or when they are responding to a local infection produce RNA that encodes the proteins they will use to fight off the virus. Profiling the changes in the RNA in a particular sample gives us a snapshot of how the immune system is working. We used an approach called RNA-Seq which captures all of the RNA in a sample and measures how many copies of each gene have been expressed. When two samples are compared side by side, the relative amounts of RNA can be evaluated; this is then presented as differentially expressed genes (DEG for short). The idea being that genes that change in amount are the ones that are important for the response to the infection.

Overall we evaluated 209 samples. When we pooled the data, we saw a clear increase in immune system genes following infection. This was not unexpected, the question we wanted to address was whether they were beneficial. The immune system has a dual role in disease, it is vital to protect us against viruses, but sometimes it overshoots and being in in excess of that required to clear the virus it can damage the lungs and cause us to feel sick. Within the data from this study, we observed increases in genes from the interleukin 17 family (IL-17). This is a type of signalling molecule called a cytokine which shapes the flavour of the immune response. It triggers a cascade of other genes, one of which is called S100A. Both IL-17 and S100A have been shown to cause enhanced disease following other viral infections, but their role in RSV is not known. Returning to the mouse model, we were able to block the action of S100A and show that when inhibited, there is less disease. Overall the study showed that integrating different data sets provides new insight and may ultimately lead to new treatments.

Bacterial Blockers

 

RNA vaccines have been the breakthrough vaccine technology of the past 5 years. They have been extremely successful in preventing disease following viral infection – particularly against SARS-CoV-2, the virus that causes COVID-19. They have also been licensed for use against another virus, RSV.

However, some questions remain as to whether they can be used against bacterial infections. One of the major questions relates to the way in which RNA vaccines work. The power of RNA vaccines is that they use the common building block of life (RNA), which is universal across all organisms. Because the platform uses the same stock material, the same manufacturing approach can be used to make any vaccine from Anthrax to Zika. The neat trick is that we are relying upon the injected cells to turn the RNA into protein, in a process called translation. However, whilst the code is the same for all living things, translating the letters of the RNA into the letters of amino acid isn’t all that is required to make a functional protein. There are subsequent modifications for example adding sugar molecules. These modifications differ between the different kingdoms, in particular, bacteria use a very different system to modify their proteins than humans. The questions is therefore whether RNA encoding bacterial genes would make properly folded proteins in human cells.

This is an important question because many of the most dangerous pathogens we face are bacterial in origin. This threat is particularly concerning due to the rise of antibiotic resistance. Which is where the frontline drugs we use to treat bacterial infections begin to fail. This is especially a concern in low income countries where rates of extremely antibiotic resistant bacteria are dramatically rising. We were interested in developing a vaccine against a bacteria called Acinetobacter baumannii which causes severe infection in people who have had to have mechanical ventilation, for example following surgery. In Vietnam, strains of this bacteria have been isolated that are resistant to nearly every single antibiotic.

An additional challenge for RNA vaccines for bacteria is choosing thee right part of the bacteria to target. Viruses are (often) simpler organisms encoding fewer proteins overall, so selecting the right one is (relatively) more simple. Bacteria can encode several hundred different proteins, which are often invisible to the human immune system.

In our recent paper, Intranasal delivery of mRNA expressing newly identified Acinetobacter baumannii antigens protects against bacterial lung disease we set out to identify and test a new vaccine targeting this tricksy bacteria. In a previous study we had used another vaccine approach called outer membrane vesicles (OMV). These are little packages that the bacteria spit out, containing a mix of proteins and other biochemicals. We had demonstrated that these could be used to protect against infection. Because OMV contain some, but not all of the proteins that A. baumannii encodes, we reasoned that some of them must protect against infection. We took two approaches to sift the proteins and found a subset of three that were in both datasets, we then generated RNA vaccine constructs of these and tested them. We were able to demonstrate protection against infection with one of the genes – Oxa23. Protection was even better if we delivered the RNA to the lungs.

We have been supported to Bactivac to undertake this work. They are a funding agency with a remit to develop new vaccines against antibiotic resistant bacteria. This is an important first step in the development of a vaccine against A. baumannii.

A scientist in a historian’s field

 On the 23^rd of June, I will take the stage (with my fellow OneWorld author, Dr Alanna Skuse) at the Chalke Valley History Festival. We are talking about ageing (the history and the biology of it).

I’ll be honest with you, this is all quite a surprise (to me at least – Alanna is a proper historian, with letters after her name to prove it). I am an academic scientist, my lab works on viral infections and vaccines – it is all on paper a long way from history.

But I have been incredibly fortunate over the last 5 years to properly rekindle a love of history. In amongst the chaos of the lockdowns, one thing that the temporary closure of my lab allowed me to do was to step back and write about the history of the prevention of infectious disease. This was a joy – I got to supplement my understanding of the immunological mechanisms of vaccines with the stories of the people who discovered and developed them. There were some extraordinary characters including Felix d’Hérelle who discovered a family of viruses that can infect bacteria, called bacteriophage. When he wasn’t doing science, he was busy living close to the pivotal moments of the 20^th century. Amongst other things, he lost all his money in a failed chocolate factory, worked in Guatemala, Mexico, France, India, the USA and Egypt (not trivial before international flight), had a brief run in with the secret police in Soviet Russia when his mentor fell in love with the same woman as Beria (the notorious head of the NKVD) and was put under house arrest by the Wehrmacht in the Second World War.

One thing I became really grateful for were the academics who had painstakingly put together the histories of medicine. For example, Louis Miller and Xinzhuan Su who pieced together the story of the discovery of the anti-malarial drug artemisinin and the role of Youyou Tu from the fragmented records of the cultural revolution. When I was a young scientific trainee, I was much too focussed on where the science was going to care about where it came from.

But writing Infectious and my new book Live Forever has given me a chance to reflect on the similarities between science and history. Both of them are trying to piece together a narrative with incomplete pieces, just in different directions – science faces forwards, history backwards. And new discoveries can change the narrative. I was discussing this with a contemporary from university who went on to become a history academic. And we turned to the role of the narrator in history and how current academic thinking has built in the narrator’s bias into the process; in science we have systems where we aim for objectivity, but there is still a role for narration and therefore bias. One of the problems of the education system in the UK is that it funnels children into silos – arts or sciences; and you lose some of the interplay between the two. And whilst I was lucky enough to be able to take A level history alongside my science subjects, there was a long fallow period before I returned to it (though my interest never faded – as can be attested by my towering to be read pile). One of my proudest parenting moments was when my son, who also splits STEM/ history told me that whilst he wanted to do a science job, he was inspired by me to carry on history on the side (you can actually have your periodic table and re-enact battles on it too).

Over the past 5 years, I have very much rekindled my flame of interest for history – particularly for WW2, and it has enriched my life by meeting others with similar passions (some might even say afflictions). I look forward to meeting you there!

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 The cost of doing treatments to live longer is extraordinarily expensive. One of the ultra-low calorie diets I tried cost me £180. 

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On birthdays

Today finds me questioning why we celebrate each complete circuit around the Sun. Something we can no more control than the day we were actually born. And yet so much fuss is made of it. I’ll admit to being much more at the Eyeore end of the spectrum, though I do hope to receive more than a burst balloon and an empty honey jar.

It doesn’t help that my big day is no longer as big as once it seemed. I’ve had a bit of a strange run of birthdays in recent years. My wife was away for two of the last three; and whilst I am airing grudges, I still am getting mileage about my parents not taking me out of school on my 10^th birthday 3 ½ decades ago. The other thing that doesn’t help is that you are expected to work and do all the normal adulting stuff.

One aspect that troubles me less is the age. Whilst the number ticks up on the odometer of my life with each passing year, it’s just a number. It’s unavoidable. This attitude may seem odd given I have just written a book about ageing and death. The central conceit of which is that I have somehow passed a tipping point from youth into middle age.

But one of the things that researching Live Forever made me realise is that since time is passing and there is nothing we can really do to reverse the onset of ageing there is very little point in worrying about it.

So, where does this leave me? Well geographically I found myself on my own exploring Kraków, the day after sitting in a slightly too hot room, reviewing scienece with an eminent professor from the Baltics who for some reason chose to remove his shoes for the duration. For once though, I had built in enough time to do something other than the usual Airport-Novotel-Conference Centre-Airport-Home. But what I didn’t quite succeed this time was to persuade anyone else to up sticks and to join me. And whilst I would like to visit it, the idea of touring a concentration camp on my own as I enter my late-late 40s was a bit too bleak.

So instead I packed my running shoes and am just back from slowly ambling along the Vistula, pondering events that happened merely 30 years before my birth and feel more worryingly close now than they have in the last 30, which in it’s own way is no less bleak. But let’s not end on that dark note. On the flight here, I treated myself to an Easyjet meal deal, ending up with an empty Pringles pot in which to store any burst balloons I acquire. Eyeore would no doubt be quite envious.