Professor Robin Shattock speaking with Research Assistant Hadijatou Sallah at our St Mary’s Campus during the development of the COVID-19 vaccine.
Since the beginning of the pandemic, researchers across Imperial have been working overtime to understand the SARS-CoV-2 virus and to develop treatments for COVID-19, the disease it causes. Animal research has been crucial to this effort, from testing candidate vaccines to revealing how and why the virus has such a devastating effect on our health.
The challenge of developing a vaccine against SARS-CoV-2 was taken up early on in the pandemic by Professor Robin Shattock, from the Department of Medicine. His group was already developing a novel vaccine platform for use against diseases such as Ebola, Lassa fever and rabies, which could be adapted for the new virus. The platform works by delivering strands of genetic code, called RNA, to cells in the body, where they produce a protein usually found on the surface of the virus in question. This protein is recognised by the body’s immune system as foreign, priming the system to react if it later encounters the virus.
The novelty in Robin’s approach is that the RNA works by making many copies of itself within the body’s cells. The result is that only a small amount of this self-amplifying RNA is required to produce an effective immune response, which should ultimately make it cheaper and easier to produce vaccines in large quantities.
Having selected the SARS-CoV-2 spike protein as a suitable basis for the vaccine, it was relatively simple to cut and paste the necessary genetic sequence into a version of the vaccine platform designed for Ebola. The RNA produced in this way must then be wrapped in a tiny droplet of fat, which helps deliver it to the target cells in the body. Formulating this droplet so that it works effectively is not so simple, and animal research is required to see that an immune response is indeed being produced.
“Ours are the simplest and mildest of animal experiments,” explains Dr Paul McKay, who worked on the project. “We inject the animals twice, and take blood to see if there is an immune response. Then, the animals are culled to harvest the spleen and other organs to examine the immune response in detail.”
As much preparatory work as possible is done in cell culture, to reduce the number of animals used, but it is impossible to simulate the complexity of the body’s immune system in a dish. “There is no replacement for an intact immune response in an animal.”
A range of animals was used in this work, beginning with the lowest vertebrate species possible. “We did the initial assessment in mice, and saw that the vaccine worked extremely well,” Paul says. This was followed by studies in rats, to assess the toxicology of the vaccine in preparation for a human clinical trial.
Smaller studies were also done in hamsters, ferrets, rabbits, guinea pigs, pigs and monkeys, either to answer specific questions about the vaccine or to see if additional data could be gathered. As always, animals were only used when a question could not be answered in any other way, and the type of animal used was considered carefully. The work with monkeys, which did not take place in Imperial’s animal houses, was a challenge study to see if the vaccine gave them practical protection when they were exposed to SARS-CoV-2. Such studies could not, at that time, be carried out with people, and the other animal models were not close enough to humans to produce a meaningful result. Once animal trials had been completed successfully, the researchers were able to move forward with stage I and II clinical trials in humans.
This research will enable us to develop, perhaps, a vaccine booster that is effective against several of the variants.”
– Dr Paul McKay, Faculty of Medicine, Department of Infectious Disease
While the Imperial vaccine produced an encouraging immune response, others using more established technologies were further along in their development, and a number of effective vaccines began to be rolled out globally. So Professor Shattock’s group has moved on to explore whether the Imperial vaccine can be adapted for use against new variants of SARS- CoV-2. “This research will enable us to develop, perhaps, a vaccine booster that is effective against several of the variants,” says Dr McKay. And since the formulation for delivering the vaccine will remain the same, the initial animal studies will not need to be repeated.
What the group has learned in the last year will also have benefits beyond the present pandemic, given the technology’s potential for use in vaccines against many other diseases. “We’ve developed a lot of knowledge and knowhow around the self- amplifying RNA vaccine platform, which otherwise might have taken us five to ten years to accomplish. It has really accelerated our research.”
Tracking virus transmission
In parallel with efforts to develop a vaccine, researchers at Imperial have also been investigating how the SARS-CoV-2 virus is spreading. This was just a small sideways step for Professor Wendy Barclay and her group, who had been asking similar questions about pandemic influenza when SARS-CoV-2 emerged. “We’ve been trying to understand how pandemics arise, with a particular focus on transmission between individuals. Transmission is key to sustaining outbreaks,” says Wendy, who is the Head of the Department of Infectious Disease.
Part of her group’s previous research had involved studying how the flu virus passes between ferrets. While relatively unusual as a laboratory animal, ferrets are a good model for studying flu because they are infected by the virus and develop symptoms in a similar way to people, whereas mice and rats do not. When the COVID-19 pandemic set in, Wendy decided to find out if her ferrets were also a good model for the transmission of SARS-CoV-2.
This could be done relatively quickly because the influenza procedures put on hold were already at the right biosafety level for working with SARS-CoV-2. The main technical challenge involved the way the samples collected from the ferrets were analysed. “We were working with a new virus, which grows in different cells and has different characteristics, and we needed to make sure that everything lined up.”
The group decided to focus on a particular sequence – a furin cleavage site – in the SARS-CoV-2 spike protein, the part of the virus that helps it infect lung cells. “This sequence rang so many bells for us,” Wendy explains. A furin cleavage site features in some highly dangerous bird flus, yet it is not present in SARS-CoV-1, which caused more easily controlled disease outbreaks in 2003. “So, we wanted to test the role of the furin cleavage site in infection and transmission in the animal model.”
This was done by comparing a circulating strain of SARS-CoV-2 and a variant that lacked the furin cleavage site. Ferrets could be infected with both kinds of virus, but the version without the furin cleavage site proved much less transmissible. Only low levels were breathed out by infected animals, and healthy ferrets housed alongside did not become infected. With the circulating virus, exhaled levels were high and neighbouring animals fell ill.
Together with cell culture studies and observations of variants in the human population, this work suggested that the furin cleavage site is indeed crucial for transmission. Knowing this helps scientists assess the risks posed by new variants of the virus as they emerge. “When we look through the thousands of sequences that are being reported, if we see one that has an optimised furin cleavage site then that is a clear warning signal.”
While the ferrets proved to be a good way of testing the transmission of SARS-CoV-2, they are not the best model for further research, because they do not become ill when infected with the virus. So Wendy’s group has now switched to golden hamsters. “They get quite sick, and are a much better model of the moderate to severe end of the COVID spectrum in people.”
Switching to hamsters has meant adapting the group’s research methods, but also updating licenses, carrying out new safety assessments, and building appropriate facilities for the new work. “A whole team of people has helped and advised us, allowing us to scale up quickly,” Wendy says. “Everyone has pulled together. That has been a real moment of pride for all of us.” Meanwhile the approach to the 3Rs developed for the ferrets has been carried over to the hamsters. This includes keeping infection levels to a minimum, so that the animals do not suffer severe symptoms unnecessarily, and adapting nasal washing techniques that allow virus levels to be assessed without distressing the animals.
But new approaches are also being developed, such as a method for studying SARS-CoV-2 in the lungs that does not involve killing and dissecting the animals. This makes use of genetically modified versions of the virus that give off light, which can be detected in live animals using sensitive imaging systems. “That means we will be able to watch the virus move, over time, between the upper and lower respiratory tracts, in the same animal,” Wendy says. This will reveal where the virus is when the animals become sick, and when transmission begins. “That will tell us a huge amount. It also means that we use many fewer animals to see where the virus is within the body.”
Having good animal models, which we have completely characterised, can be really useful to drive the basic understanding of the disease and work on therapeutic targets.”
– Dr Cecilia Johansson, Reader in Respiratory Immunology, National Heart and Lung Institute
Explaining the severity of infection
Just what happens when SARS-CoV-2 reaches the lungs is a question that Dr Cecilia Johansson turned to when the pandemic broke out – a question she was able to investigate using mice. Cecilia is no stranger to respiratory viruses, having worked for many years on respiratory syncytial virus (RSV) and influenza, in particular studying the immune responses they generate in the lower airways. “It was obviously very interesting to ask the same questions about the new SARS-CoV-2 virus,” says Cecilia, from the National Heart and Lung Institute.
These questions are almost impossible to answer by studying people, and not just because it is hard to sample human lung tissue. “We are interested in the early events and how inflammation is initiated. That happens a long time before someone arrives in hospital with signs of the disease.” Animal models make it possible to follow the disease from the very first moments of infection.
The first challenge for Cecilia was that the mice she usually works with are not susceptible to the circulating strains of SARS-CoV-2. Various approaches are now being tried to create a useful model, either by genetically modifying the mice to make their cells appear more human, or by modifying the virus so that it targets the mice more effectively. The important point is not so much that the mice should have the same symptoms as people, as the same kind of immune response when the virus arrives in the lungs.
“In a lot of cases, including COVID-19, the virus infection is cleared from the lungs, and what we suffer from is an uncontrolled immune response, which causes a lot of damage,” Cecilia explains. “So, in the mouse models we want to see the same kind of immune response, with the same mediators produced and the same cells coming in, so that we can find out where this regulation of the immune response goes wrong.
Once a mouse model has been validated as a good representation of what is happening in humans, more in-depth characterisation of the immune response and lung damage can begin, using the techniques she and her colleagues have developed for other viruses. One of these involves taking thin slices from the lungs of the mice, which can then be used to answer a range of different questions, maximising the data gathered from each animal. “We are developing that method in parallel with the model validation work, in order to use fewer mice.”
The main aim of this research work is to increase our basic understanding of how SARS-CoV-2 operates. “The big question in my field is: why do some people get really sick, and in the case of SARS-CoV-2 even die, while other people are asymptomatic and don’t even notice that they have the same infection. So, what is driving the severity of the disease?”
Revealing this mechanism will help improve assessments of individual susceptibility to the disease, and suggest targets for new treatments. And the animal models themselves will be tremendously important in other research. “Having good animal models, which we have completely characterised, can be really useful to drive the basic understanding of the disease and work on therapeutic targets.”
Looking back on the past year, Cecilia saw a unity of purpose in this work on COVID-19, not just among the researchers at Imperial, but also the administrators, the technicians who care for the animals, and other support staff. “This disease has had an impact on everyone, and everyone wants to contribute.