A Pre-Pandemic vaccine.

 A paper about pandemic vaccines: shaped by a pandemic.

We first started writing the grant application that supported this work in 2017. The work was funded by the Coalition for Epidemic Preparedness Innovations (or CEPI). CEPI is a global partnership that was set up to accelerate the development and delivery of vaccines. The original plan was to look at three viruses – the one I was leading was influenza. We had a remit to develop a vaccine that could provide protection against infection within 6 weeks of the first immunisation.

The approach we used was an RNA vaccine, which unless you have been living under a rock for the last 3 years I am assuming you have heard of. But just in case, what these vaccines do is to take the genetic material that encodes a tiny bit of the virus and inject it into the muscle. Once injected your muscle cells make the viral protein training your immune system to recognise them. This means that when you are exposed to the real virus, you can fight it off better. Specifically we were using a self-amplifying RNA vaccine, this is subtly different; you can in theory get bigger responses for smaller amounts of material. I have described them before here.

One of the first questions we looked at was how best to formulate the vaccine; the work is described in our recently published paper Formulation, inflammation and RNA sensing impact the immunogenicity of self-amplifying RNA vaccines. RNA is quite unstable and needs to be mixed with other compounds in order to get it into cells. We tried three approaches a cationic polymer (pABOL), a lipid emulsion (nano-structured lipid carrier, NLC) and three lipid nanoparticles (LNP). In simple terms to get RNA into a cell, you either need to add positive molecules (the cationic polymer) or some fat bubbles (the LNP and the NLC). We noticed that responses to the LNP were very much better than the other approaches and wanted to understand why.

One of the questions we asked was about the role of inflammation. Inflammation is a cascade of signalling by which the immune system recruits cells to a site of infection or danger. In conventional vaccines it is important because it alerts the cells that there is something foreign to be recognised and trains up the vaccine response. However, there was a question about its role in RNA vaccines, because RNA vaccines need to be made into proteins in the body to work some aspects of the immune system might inhibit this. Surprisingly (which is up there in the go to words of academic writing, alongside interestingly), we saw that vaccine induced inflammation was associated with better, not worse responses. The next step is to further dissect how this inflammation is beneficial.

Coming back to the timing of this project. We started the labwork in 2019, when the idea of a world changing pandemic virus was somewhere in the future. Then of course the events of 2020 caught up with us. The work definitely slowed down – though aspects of it were incorporated into the Imperial College vaccine trial. The formulation that we had showed to work best in the saRNA system was the one used in the clinical trial. As part of the ongoing vaccine research, I was able to come into the lab occasionally, providing a sanity lifeline and also the very odd experience of commuting through an empty London. Where this exciting vaccine technology goes next is still an important research question and one I look forward to continuing to investigate.

Do dirty viruses make you sicker?

 I think we can all safely agree that the immune system is complicated; that viruses are complicated and when you combine the two the overall outcome is more complicated than the sum of its parts. In the current study (Levels of Influenza A Virus Defective Viral Genomes Determine Pathogenesis in the BALB/c Mouse Model) led by Becky Penn in Wendy Barclay’s group at Imperial College London, the question was what happens when the infectious virus (in this study influenza) contains more (or less) junk.

Viruses contain genetic information wrapped up in a protein coat. For a more detailed overview of what viruses are – can I recommend my book Infectious (out now in paperback). The immune system recognises viruses through a number of different ways, but one of them is through recognising the viral genetic material. In the case of influenza this is somewhat more simple because the genetic material is different to human genetic material. Humans pass their genes between different generations using DNA, but influenza uses a related molecule called RNA. Human cells also use RNA, but for different purposes – mostly for transmitting genetic information within the cell. The viral RNA therefore is a clear danger sign to cells that there is an infection. Viruses have evolved to hide their RNA from the immune system. They wrap it around proteins like thread wrapped around a cotton reel. This means the immune system can no longer see the RNA and reduces the response to it.

However, the amount of RNA is not constant. Sometimes individual virus particles fail to hide their RNA, which makes the immune response to them stronger. One factor that affects the packaging of the RNA is how the viruses grow. Becky manipulated the way that she grew her influenza virus, with some of the stocks having much more of these badly packaged viruses and some having neatly packaged genetic material. The theory was that the badly packaged viruses was, because they are more dirty trigger the immune response, what was unknown was how this would effect the outcome. There were 2 possibilities:

1. The immune system would be so fast at seeing the virus, it would kill it before it ever took hold.
2. The immune system would go into overdrive.

Surprisingly, the results were a bit of both. The dirty viruses (also called High DVG because they have more defective viral genomes) were indeed detected quicker by the immune system. This led to the release of signalling molecules called interferon. Interferons trigger an anti-viral state, shutting down the growth of virus within the cells.

The cleaner stocks performed very differently. They were initially able to escape the immune system and set up an infection in the lungs. This infection then, paradoxically, led to the production of the dirty viruses in the lungs of the infected animals. This production in the lungs led to a cycle of inflammation, basically putting fuel on the fire and increasing the severity of disease. This tells us that disease after viral infection is driven by a combination of factors – the damage caused by the virus itself and the immune response to the damage. On a more niche note, it tells us that the quality of experimental virus used is really important in shaping the outcome of the study. Which is one of the almost infinitely complicated variables in doing biological studies with 2 live agents (let’s not even begin talking about time of day, chronobiology and the impact that has!

Infectious now in paperback

 

My book is now out in paperback - available wherever you buy books

Can we save lives by deliberately infecting people?

In the middle of the pandemic, scientists intentionally infected healthy volunteers with SARS-CoV-2, the virus that causes COVID-19. John Tregoning, Reader in Respiratory Infections at the Department of Infectious Disease, explains why these experiments, and the volunteers who take part in them, are critical to modern medicine.

In early March 2021, in the middle of the COVID-19 pandemic, a surprising-sounding set of experiments were taking place. Researchers at Imperial College London (and separately at the University of Oxford) were deliberately infecting healthy volunteers with SARS-CoV-2. This was in fact the latest in a long line of controlled human infection studies – where volunteers are deliberately infected with an infectious pathogen under extremely controlled conditions.

Deliberate human infection for health benefit goes back a long way – the earliest evidence of infection for beneficial use is 10th Century China, deliberately inoculating healthy people with smallpox to make them immune to the disease. This practice continued into the 18th century, when an English doctor, Thomas Dimsdale, deliberately infected Catherine the Great and her son with a very low dose of smallpox virus to protect them against the disease.

This idea of infecting people deliberately to protect them from disease led to Edward Jenner’s famous studies inventing the first ever vaccine. Jenner hypothesised that you didn’t need to use material derived from smallpox to be protected, you could use material from a related virus, cowpox. He proved this worked using a human challenge study; he vaccinated James Phipps (his gardener’s son) with cowpox then deliberately exposed him to smallpox repeatedly, showing that the vaccine worked and Phipps was immune to smallpox.

Deliberate infection

The practice of deliberate infection for scientific benefit really took off after the demonstration by Pasteur, Koch and others that microbes cause disease. In the early 1900’s, Walter Reed, the American public health pioneer, was trying to understand where yellow fever came from – he had a suspicion that it came from mosquitos. This was important because identifying the source could alter behaviour and reduce the incidence. To test his hypothesis, Reed recruited 11 volunteers to be bitten by mosquitos that had previously bitten a yellow fever patient; two of the volunteers contracted yellow fever, strongly supporting his idea. One important development in Reed’s infection studies was the concept of ‘informed consent’. The volunteers were informed about the risk to themselves of participation, before they gave their consent to take part. Sadly, later in the 20th century, some human infection studies entered a darker chapter, with the horrific experimentation on prisoners in Nazi Germany and Imperial Japan without their consent.

Informed consent is the bedrock upon which all modern research involving volunteers is built, and it is crucial for infection studies. The landscape of human infection studies has changed dramatically since the middle of the 20th century; now, ensuring the health and safety of participants is of paramount importance and trials are carefully designed to minimise any potential risks. Studies are only performed following extensive ethical review by an external body, for example all human infection studies carried out at Imperial College London have ethical approval from the UK Health Research Authority. There is ongoing debate about whether infection studies can ever be ethical, in terms of deliberately exposing someone to the risk of harm; even in the context of minimising the risk. However, there are many benefits to the studies and when volunteers understand the risk and choose to participate for the greater good, they can achieve important things.

Vaccines

One of the ways in which deliberate human infection studies (or ‘challenge studies’) are most beneficial is in the testing of vaccines. Vaccines are tested in the same way as any drug and the first studies involve a small number of participants who receive a dose of vaccine and are closely monitored to check first and foremost whether the vaccines are safe. These early studies (called Phase I clinical trials) can also inform about whether the vaccine is making an immune response. However, in order to demonstrate that the vaccine can prevent disease, much larger studies are needed. These studies (phase III) are often very large, because you can’t be sure how many of your vaccinated volunteers will then be exposed to the infectious agent. This means that you need huge numbers of subjects to get to statistically meaningful numbers to compare infection rates with and without a vaccine.

Infectious challenge studies can help to overcome this barrier, especially when the pathogen being tested is rare. One example of this is typhoid, a bacterial infection that causes diarrhoea in approximately 10-20 million people a year, mostly in low- and middle-income countries. A research team in Oxford gave volunteers a typhoid infection and tracked them untill they had clinical symptoms before treating them with antibiotics. Again, pausing to think of the volunteers – knowing that you are likely to get a bout of diarrhoea and going ahead with it anyway, for other people’s benefit, takes a special mindset.

Indeed, without volunteers, modern medicine would falter, so we all owe a large debt of thanks to these selfless individuals. Having shown it was possible to infect people in a controlled way, the Oxford group tested whether two new vaccines could reduce disease. They showed that whilst 77% of the volunteers without a vaccine developed typhoid, only 35% of the vaccinated volunteers did. Deliberate infection studies have also been used to support the rollout of vaccines for cholera, malaria and shigella.

Measuring antibodies

Another important benefit of deliberate human infection studies is in understanding how specific viruses cause disease and how we can be protected against them. The Common Cold Unit was a British research centre operating on Salisbury plain between 1946 and 1989. It set out to understand respiratory infections; being somewhat isolated it was able to look at transmission of colds, by infecting one volunteer and then housing them together with other uninfected volunteers. It also provided us with important information about the levels of immunity required to protect against influenza. By measuring antibodies in the blood of people before they were infected it was possible to identify a threshold above which infection was unlikely to occur; this threshold is still in use for the development of influenza vaccines.

Some diseases have more challenges than others in setting up the infections. Whilst respiratory viruses can be grown and dripped into the nose, other infections get into our bodies through a third organism, called a vector. In the case of schistosomiasis (sometimes called bilharzia), the disease-causing parasites live in snails (the vector) before infecting people. To help develop drugs and vaccines for this neglected tropical disease, researchers have had to learn snail husbandry!

Returning to the coronavirus human challenge studies, these look to address both the development of vaccines and improve our understanding about infection. In the earliest results from the Imperial-led study, the team showed symptoms start to develop very fast, on average about two days after contact with the virus. The infection first appears in the throat; infectious virus peaks about five days into infection and, at that stage, is significantly more abundant in the nose than the throat. It was seen that volunteers who had not had COVID-19 before could be infected with an extremely low dose of the virus, which might help to explain why SARS-CoV-2 is so infectious. It can also inform more generally about the behaviour of respiratory viruses. These studies are now progressing to help in the design and testing of the next generation of vaccines and drugs.

As we have seen in the last two years, infections can be extraordinarily disruptive. Studying how they behave, why we get infected and how to prevent this is extremely important – when performed safely and ethically, human infection studies are an important part of our toolkit.

 This first appeared on the Imperial College, Faculty of Medicine Blog

Ferreting about

 

As has been clearly shown during the COVID-19 pandemic, RNA vaccines are an incredibly powerful tool. We have seen how potent they are against a rapidly emerging infection, with the speed at which they can be produced a major benefit. From the first genome sequence published to the first experimental vaccine dose designed, manufactured and administered was seventy-one days, less time than it takes to make cheese. However, more work is needed to deliver on the promising start that RNA vaccines have had.

One of the challenges has been with the side effects of the vaccine, sometimes called reactogenicity. This is in part caused by the body’s reaction to the vaccine itself: RNA is a trigger for immune reactions, it is how the body sees viral infections. Tweaking the amount of RNA in the vaccine, whilst maintaining its ability to induce a protective antibody response is one way in which we can improve RNA vaccines.

We have been interested in a slightly different approach, called self-amplifying vaccines. We showed (pre-pandemic) that these saRNA vaccines can lead to protection for a much lower dose. These vaccines are based on RNA, but able to make more copies of themselves in the injected muscle, giving you more bang for your buck. Professor Robin Shattock (also at Imperial College London) performed the first in human clinical study using saRNA vaccines during the pandemic. This trial had a mixed result, with not all of the volunteers producing antibodies after vaccination. In parallel with this first in human study, we looked at how the vaccine might work in other model organisms, as a way to understand how these vaccines work.

In our recent study Polymer Formulated Self-Amplifying RNA Vaccine is Partially Protective against Influenza Virus Infection in Ferrets, we tested the immune response to saRNA vaccines in ferrets. This might sound like an odd choice, but ferrets have a number of similarities to humans in they way they respond to infections, especially influenza. We set out to test whether saRNA vaccines could protect ferrets against flu. As with the human study, we had mixed results. The ferrets that responded to the vaccine by making antibody were well protected against infection, however, not all of the ferrets made antibodies. Understanding why this is the case is important in the future development of this extremely promising vaccine platform.

Ever shifting strains

The bacteria Streptococcus agalactiae is more commonly known as Group B Streptococcus or GBS. The Streptococcus groupings were developed by Rebecca Lancefield in the 1930s. They are somewhat redundant now due to other methods of categorising bacteria, but Group A Strep and Group B Strep are still commonly used (Group A strep or GAS is Streptococcus pyogenes, which causes a range of diseases, the most well known of which is scarlet fever).

Anyway back to GBS, which is mostly harmless – until it isn’t. It can colonise people without causing an infection. It mainly lives in the gastro-intestinal (GI) and genito-urinary (GU) tracts. Unfortunately, it can have major and severe impacts during pregnancy. It is the leading cause of neonatal infection, having severe consequences for the newborn baby. It passes from the mother, who has some level of immune protection, to the baby (who doesn’t) during, or shortly after, birth. Whilst antibiotics can reduce the burden of disease, they are not completely effective and alternative preventative approaches are needed. One of which is vaccination, but one of the challenges is that the mechanism by which women are protected against disease is not well understood. More work is needed to understand the interplay between the immune system and the colonising bacteria.

In our latest study, Group B Streptococcus (GBS) colonisation is dynamic over time, whilst GBS capsular polysaccharides-specific antibody remains stable we worked with Professor Kirsty le Doare and her group at St George’s University in London to look at the interplay of GBS colonisation and the immune response. Women were recruited and had samples taken from their GI-GU tracts every 2 weeks for 12 weeks. We then measured whether GBS was present, which strain of GBS it was and whether colonisation led to an increase in the amount of antibody the women made.

We observed that colonisation with GBS was dynamic – there were volunteers that were colonised initially that subsequently cleared the colonisation, there were others that acquired bacterial colonisation, there were some that were never colonised and others that had multiple different strains. However, whilst the bacterial colonisation was variable, the levels of antibody were fairly constant implying that acquisition of bacteria may not be directly affecting whether the women make more antibody – or that they have previously been exposed to the same strains and therefore don’t make new responses. Alternatively, there may be something special about the guts that mean that bacteria that live there (but are not necessarily causing disease) don’t trigger the immune response.

This study was performed during the COVID lockdowns which significantly disrupted the collection of samples. However, thanks to innovations by the study leads – such as home sampling, it was possible to continue. These kinds of approaches may mean larger studies can be performed as they are less intensive on the researchers and participants.

This study demonstrated the complex interplay between host and bacteria. Further investigation is needed to understand how best to utilise a vaccine to protect against this devastating disease.

Don't T me off

 

Vaccines can be made of all sorts of things – the pathogen itself, either killed or in a weakened form; protein or sugar from the coat of the pathogen or nucleic acid derived from the pathogen. This nucleic acid can either be double stranded DNA or single stranded RNA. And whilst RNA has suddenly acquired celebrity status, the DNA based approach is the older (slightly less successful) sibling.

One of the ongoing challenges with nucleic acid-based vaccines is balancing the expression of the vaccine antigen against recruiting the immune cells that are needed for the vaccine response. This is because, unlike older vaccines, nucleic acid vaccines need the cells of the body to make the protein that the immune system will recognise in situ. There are 2 challenges with this, firstly the DNA itself can trigger a cascade of events that mean the cells are less likely to make the DNA encoded protein and secondly, if the cells can be persuaded to make the foreign protein, it can act as a flag for the immune system to target and attack those cells.

We were interested in this second challenge in our recent study: Blocking T-cell egress with FTY720 extends DNA vaccine expression but reduces immunogenicity. We wanted to understand how one arm of the immune system – the T cell impacts the response to DNA vaccines. T cells have a range of roles, including where one T cell subset (CD4 T helper cells) orchestrates the immune response to another T cell subset (CD8 T killer cells) to facilitate killing of infected cells. The CD8 cells recognise target cells because infected cells wave tiny bits of foreign protein on their cell surface.

To explore the role of these different T cells in the context of DNA vaccines, we made use of a protein called luciferase; this comes from fireflies and gives them the ability to produce light. Technically speaking it is an enzyme that catalyses the breakdown of a molecule called luciferin, when luciferin is chopped up by luciferase it becomes unstable and can only relax by releasing light energy in the form of light.

We injected DNA encoding luciferase into the leg muscle of mice and measured how much light their legs emitted. As expected, the injected mice produced light (not loads – you need a special machine to measure it); the amount of light peaked 2 days after injection but disappeared 3 weeks later. To test the role of T cells, we gave one group a drug called FTY720 (or fingolimod, which is why we refer to it as FTY720). FTY720 has the interesting property of preventing T cells moving around the body – so they can’t get to where the luciferase was being made. The mice treated with FTY720 kept on producing light for as long as we injected them with the drug. The FTY720 injections could be staggered, it just needed to be kept over a threshold.

We thought this was then going to be good news in terms of the immune response, reasoning that the longer the vaccine was made in the cells the greater the response. So, we repeated our study, but instead of using DNA making luciferase and measuring light, we used DNA that encoded the surface protein from HIV. In this experiment, the FTY720 significantly reduced the immune response to the vaccine – with the treated mice making much less antibody.

These studies reflect the fact that T cells have 2 roles; the killer CD8 T cells which can kill the cells that have taken up the DNA vaccine and are making the foreign protein but also the helper CD4 T cells which boost the immune response. FTY720 was a blunt instrument shutting down both types, with a resultant negative impact. This study showed us that T cells can limit expression of DNA vaccines in the host cell, but we need to do more research to extend that expression without impacting other arms of the immune response.