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.

The complex world of the child’s nose.

As any of you who have children, in fact anyone who has ever met, seen or been a child, there are all sorts of things up their noses, fingers, Lego pieces, but more importantly viruses. They are awash with different viruses. We are particularly interested in the effect of one viral infection on another.

We investigated this in the context of a vaccine study. There is an influenza vaccine that is live, but genetically weakened; called live attenuated influenza vaccine or LAIV. This vaccine is sprayed up the noses of children where it causes a limited infection that can train the immune system and protect against future influenza infections. This gives us a safe way to explore how flu infections happen in children and what factors affect infections particularly the immune response. This study was performed in The Gambia by Dr Thushan de Silva.

In our recent study: Prior upregulation of interferon pathways in the nasopharynx impacts viral shedding following live attenuated influenza vaccine challenge in children we looked at the influence of the presence of other infections on LAIV infection. The first thing we measured was the presence of viruses in children – remarkably 42% of children had detectable viral RNA. This reflects other studies we have done where we saw that 20% of children had another viral infection. For the analysis we then compared the responses in children with and without another infection and what impact this had on how well the LAIV viruses could establish a local infection in the nose .

In particular, we were interested changes in the genes in the nose due to infection with other viruses. These changes in genes give us a more global sense of the response to infection. What we are actually measuring are changes in RNA levels, called the transcriptome. We can group the individual genes into families based on their function. We are particularly interested in genes associated with the immune response.

What we observed in our study was that children who had another infection at the time of LAIV immunisation had far greater levels of genes associated with signalling through a pathway called type I interferons. These genes are part of the innate immune response to infection and switch on an antiviral state, which makes it more difficult for other viruses to infect them. In line with this, children with higher levels of the anti-viral genes had lower LAIV viral replication.

These findings might help us understand differences in outcome following infections such as flu and how other viral infections immediately before exposure could influence this.

On mutation and namings

Viruses are unusual; unlike other organisms which always use DNA to pass information between generations, viral genetic material isn’t always DNA. Some viruses use RNA, which human cells mostly use to transmit information from the nucleus to the machinery that makes proteins. These differences in the way in which viruses encode their genes have a profound impact on viral evolution. When human cells make copies of themselves, they use proofreading to ensure that the DNA copies are identical to the original code, this reduces the rate of mutation. However, viruses that use RNA to transmit their genes lack this proof-reading capacity, leading to a much higher mutation rate. Whilst this can be deleterious for an individual offspring virus, for the population as a whole it is extremely effective. This comes down to numbers, higher species only produce limited numbers of offspring, so each one needs to be as good possible. Viruses go for safety in numbers, really big numbers. Each infected cell produces approximately 10,000 new viruses. Therefore, there is a huge stock of different offspring, a small number of which will be fitter than their parents. This high degree of mutation means it can be tricky to apply the Linnaean system to group viruses. We can loosely group viruses on a range of different characteristics or the similarity of their genes, but because they change all the bloody time, they are hard to pin down exactly.

Viruses can be named after the disease they cause: influenza virus is named after influenza and yellow fever virus after yellow fever, this is admittedly confusing, but reflects how the disease was known before the causative agent. Alternatively, viruses are named after the part of the body they infect, in Greek to make it sound more sciencey, hence rhinovirus rather than nose virus (rhino is the Greek word for nose). Finally, viruses have been named the geographical region where they were discovered (Ebola after the Ebola river, Lassa after a village in Nigeria). The geographical naming of viruses stopped due to the stigma attached, which is why SARS-CoV-2 took three months to be named and wasn’t called Wuhan virus. Though a different approach has been used recently of using Greek letters for the SARS-CoV-2 variants of concern.

 Excerpt from Infectious: Pathogens and how we fight them

Signalling failure delays training of antibody in early life

 

This may come as a surprise, but there are other viruses that infect our lungs than COVID! One of them is called Respiratory Syncytial Virus (RSV). This innocuously named virus is THE leading cause of hospitalisation in children during winter months, but somehow doesn’t get the same level of attention as its sexier relatives influenza and SARS. One of my colleagues Prof Peter Openshaw has previously suggested it be called ‘deadly killer virus’ to get it the attention it deserves.

One of the interesting aspects about RSV is that it is possible to get re-infected with the same virus. This is unusual because the assumption is mostly that once the immune system has seen a virus once it is then better trained to deal with it in the future, preventing further infections. However, some viruses such as RSV (and also coronaviruses, like the one that causes COVID) can reinfect. We don’t fully understand why this is the case, but one contributing factor is a protein in the blood called antibody. Antibodies are highly specific molecules made by the immune system that can bind and kill viruses.

Antibody molecules are produced by a white blood cell called the B cell, but in order to produce the best possible antibody, B cells need help from another type of cell called the T cell. The conversation between T and B cells happens in the lymph nodes – which is why you get swollen glands after infection or immunisation. In our recent work, ‘Enhanced IL-2 in early life limits the development of TFH and protective antiviral immunity‘ recently published in the Journal of Experimental Medicine we explored this interaction. Specifically, we asked the question are there differences between the T-B cell crosstalk in early life – the time of greatest susceptibility to RSV.

We found that baby mice infected with RSV produce less antibody than adult mice infected with the same dose. This lack of antibody left the mice susceptible to re-infection with the virus. Side by side with the reduction of antibody, there were fewer of the helpful T cells needed to train the B cells. If we specifically removed those helpful T cells (called T follicular helper cells or Tfh) from adult mice before RSV infection, we saw a very similar effect – re-infection.

We dug deeper as to why these handy Tfh cells are not so active in early life. We identified a role for a molecule that cells use to talk to each other called interleukin two (the sequel to the commercially more successful, but less interesting interleukin one). There is more of this molecule sloshing around in early life and it may play a role in training the early immune system what is good and what is bad.

Overall, out findings may help us to develop better vaccines that work from the moment babies are born and stop them catching viruses such as RSV.

The COVID-19 vaccine effort: viruses, vaccines and variants versus efficacy, effectiveness and escape

 We tried to capture the state of play of coronavirus vaccines, it was extremely mercurial - so much data is coming out all the time. But if you want a snapshot it is here https://rdcu.be/ctIwh

In summary - all good, as long as the doses are given to people we will be ok!