Running in the same woods, twice

 Driven outside to escape the world within; the man laces his trainers, pushes thoughts of work aside and turns out of the door.

Feet hit the ground, the mud, the dirt, the puddle, occasionally the snow.

The same loop (always clockwise, less uphill that way), the same wood, the same man.

But no one ever steps in the same wood twice.

The seasons turn. Dark, cold, no leaves. Rainy, wetter, budding trees. Puffs of green, bursts of life. Days lengthen, sweat pouring. Autumn relief. Leaves falling. And back to dark, cold, bare trees.

The seasons turn again. Rutted ground becomes mud; mud becomes swamp. From concrete to impassable mire, sucking at shoes, splattering legs. In the persistent downpour, paths swell into rivers, steps become splashes. His initial reluctance to get wet lost as he plunges into the flow. A trail of destruction through the house when he returns home. But as the year moves on, water dries away, returning to mud, then back to ruts that trip and tease and turn the ankle.

And they turn again. Each time the same, each time different. Clouds, sunsets, smoke, fog. Running in darkness, his world reduced to a cone of torchlight. Isolation complete, trusting his feet to know the way. Night into twilight, murky light, daylight. Black to grey to blue. Dappled shadow, puddles of sun. Sky briefly stretching across the horizon. Solstice arriving too soon, night returns again. Immeasurably at first and then a sudden tip and his outdoor life clad in sable.

The woods dance along with the seasons. Colours change, spring lustre burnished to summer gold before autumnal shades are muted back to black. Buds to shoots to tendrils to impenetrable vegetation. Branches asleep in winter, awaken, creeping their way across the paths, whipping at eyes. Down below brambles snap at ankles, tear at clothes. His thoughts creep in too, unwelcome as the tangling vines. But he pushes on and pushes them away.

He likes to think the woods are his, but he’s not alone. Foxes bold as brass, bored of raiding bins. Skittish deer and daring squirrel. Birds whose names he knows: pigeon, duck and sparrow; so many more he recognises but cannot place: swift, buzzard, swallow. Midge and mozzie, fly and wasp. People too. Walking, cycling, riding. A nod to fellow runners. And of course their dogs. Some in control, most not. Leads tangling round legs, chasing, barking, jumping up. ‘Oh he’s friendly’ belied by scratches on legs and nips at fingers. Little presents left, some wrapped, others trapped in tread, undiscovered till shoe removed.

Senses engaged in different ways. Scentless cold giving way to spring flowers onto summer bracken then autumn decay. Brief hints of grass, notes of blackberry hanging heavy before giving way to earthier tones - a wine cellared too long.

The soundtrack on loop: silence, birdsong, dogs, people. But woodland music is overlaid with his breath. Rasping in, forced out. Effort echoed in his inhalations. His footsteps add a rhythm. Trudging, thumping, bumping, pounding. Over and over and over, hitting ground and mud and dirt. Drowning out thoughts, his perspective reduced to where the next foot goes, and the one after and the one after that.

The path and the seasons follow the same course year on year. Both stay the same; both change. This one wet; that one scorched. Weather teases tree: wind and drought tear off branches, rain washes away roots, paths are blocked, alternates opened by other feet.

The man too has seasons. The helter-skelter looping round, but twisting downwards. Aches arise, tendons twinge. Some days running is reduced to walking. What once was fast now more laboured. But some days buck the tide of time, bursts of energy restored to aching limbs. Flow achieved. Though pain awaits. The memory of pain short-lived, the pain of memory lives longer though.

The reset harder each time.

The woods draw him on: feet hitting the ground, the mud, the dirt, the puddle, sometimes the snow.

But where once unrecorded, his data is now captured, shared, liked. Freedom lost in exchange for records, badges, kudos. Virtual medals for arbitrary segments. Connections with people he’ll never meet, let alone know. Timed races against the time which ebbs away from him.

And not just the speed, distance and time but also the visuals. Favourite views that appear and disappear. Solitary oaks in cornfields. Coal posts directing long dead travellers to buildings long gone. Signage separating horse from cyclist. Visual milestones that mark his way. Once his alone, now he feels compelled to share with others. Photos perfectly cropped and curated. But in perfecting he removes all that makes perfect. Motivational slogans added, hashtags curated. The beast fed. Likes harvested from unknown users. After all, if it didn’t get captured, it didn’t happen.

Endless echoes outwards. An inescapable presence on his shoulder, driving him on. In all parts of his life. Work once contained, constrained at a locale far from home, now always on; the allure of working from home a lie, he is now never not at work. The device that lets him capture his run now capturing him. Alerts, notifications, video calls, DMs, slacks. Teams that do not play together because of bad signals, both interpersonal and electronic.

It’s enough to drive him back out. Feet hitting the ground, the mud, the dirt, the puddle and rarely the snow.

The snow falls much less often than he once remembered. The wood had also stepped tentatively onto the same helter-skelter of decline. The once reliable cycle of the seasons now as muddled as him.

The machine he feeds burns through his data and burns the air around it. His runs fuel the thing he is running from.

But he pushes this from his mind as he relaces his trainers. And turns out of the door. The same loop (always clockwise, less uphill that way), a different wood, a different man.

The other nucleic acid vaccine

 Scientists have short memories! Until 2020, the front running nucleic acid vaccine platform was DNA – it was perceived to be more stable, easier to work with, cheaper. The number of clinical trials and research spend on DNA vaccines far outweighed RNA vaccines. Then COVID came along and upturned everything. The success of the clinical trials and then the vaccine rollout ensured that RNA vaccines became a cause celebre, with much (if not all) of the vaccinology field switching platforms.

However, there is still some mileage in DNA vaccines, especially around scale up and manufacture. The problem had always been that they were poorly immunogenic, especially in humans and large animals. However, some of the technology that has been useful for RNA vaccine delivery, particularly the way in which they are formulated might assist in DNA delivery.

In our latest paper LNP-formulated dbDNA vaccines elicit durable humoral and cellular immunity with distinct kinetics to mRNA in non-human primates we explored the impact of formulation. We were working with a company called Touchlight Genetics – with whom we have collaborated for over 15 years. Touchlight have an approach for manufacturing DNA without the need for bacteria. This is advantageous because of the speed – you can go from sequence to material incredibly rapidly – vital in a pandemic.

In the study we compared a range of methods to deliver DNA vaccines. Initially we looked at the use of microneedles and a technique called electroporation, where an electric current is applied to the vaccine site which helps uptake. These worked in pigs – which are a good model of human immune response, but were maybe not as practical for use in people. So we moved to different LNP formulations to optimise which would work best with DNA. Whilst broadly similar to those used for RNA, there were some differences, relating to how the vaccine material is delivered and where it needs to get to in the cell. We compared RNA and DNA vaccines head to head, and saw an equivalent immune response, this is important as previously RNA vaccines have outperformed DNA vaccines. We then moved into larger animal models and observed that whilst the two vaccines induced equivalent antibody responses, the DNA vaccine lead to greater and sustained T cell responses.

All of which suggests there is still life in DNA vaccines and that ongoing exploration would be beneficial. The main lesson from the COVID vaccine race was that having several shots on goal is much better than banking on a single approach.

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!