A deeper picture of vaccine effectiveness
Researchers in the FuncMAB project are developing high-throughput methods to analyse the functionality induced by individual antibodies, helping to build a deeper and more resolved picture of protection on the single-antibody level, seeking answers to why people respond differently to vaccination, as Dr. Klaus Eyer explains.
The method by which the effectiveness of vaccines is assessed has remained largely unchanged for over a century. Essentially, a blood sample is taken from an individual following vaccination, and the concentration of antibodies is measured. “We check whether an individual has antibodies or not on the cellular level,” explains Klaus Eyer, a Professor in the Department of Chemistry and Applied Biosciences at ETH Zurich. As the Principal Investigator of the EU-funded FuncMAB project, Professor Eyer is now looking at this from a different angle. “We know that these antibodies are produced by cells, and that a healthy human being has around 5,000 different antibodies against various pathogens in their bloodstream. We want to analyse every antibody, by itself, for its functionality, its capacity to protect,” he outlines. “It helps here that every cell basically produces – at a given time point – only one antibody. So if we analyse individual cells, we can analyse individual antibodies, and then try to reassemble our functional analysis into a global measurement of protection and its duration.”
Vaccine protection and functional antibodies
This enhanced resolution on the level of individual antibodies could lead to the identification of signatures that allow researchers to better determine who is protected by a vaccine , and to understand why certain individuals are not protected after vaccination. While a vaccine that stimulates the production of a large quantity of antibodies is often highly effective, this is not invariably the case. “We know some vaccines that have been shown to be effective don’t actually stimulate the production of a lot of antibodies. There are also examples of vaccinations that didn’t protect, despite leading to quite a high antibody response,” explains Professor Eyer. Within the project, Professor Eyer and his colleagues are working to build a deeper picture of the antibodies produced following vaccination and their effectiveness through research conducted largely on mice for the moment to develop suitable assays and understand the basic mechanisms of antibodies. “It’s not enough simply to have antibodies, they have to do something,” he stresses. “If you are infected by a virus, you need antibodies to neutralise it. For a bacterial infection, you need to eliminate
it by activating the innate immune system using antibodies that are able to do so.”
The primary aim in the project is to develop high-throughput assays that allow researchers to measure the functionality of single antibodies in this respect. This project is not about developing a vaccine against a specific disease but rather building a deeper understanding of how the protection provided by vaccines against different diseases can be measured. “For example, for viruses, we can measure how many neutralising antibodies you generate, in relation to all the antibodies as a whole. In a further step, we can then work on increasing this proportion. For example, a simple but complicated question: How can you induce more neutralising antibodies for a longer time? It all starts being able to precisely measure the antibodies.” says Professor Eyer. “We’ve also worked on a bacterium that can infect the brain and cause an inflammatory reaction. We know that we need to have antibodies that activate the complement system against this bacteria. We can look to measure how many antibodies an individual has. Is there a way to increase the amount of antibodies to better protect individuals?”
FuncMAB project
Researchers in the FuncMAB project are developing microfluidic technologies and specific assays, hoping to open up new insights into these kinds of questions. This work involves taking individual, antibody-producing B-cells, which are then encapsulated in microdroplets.
“We use a microfluidic technique to create small containers around 50 picolitres [1012 of a litre] in size, and whatever antibodies the cell produces will stay within the small container. Within the containers, we have systems in place allowing us to measure the concentration and functionality of the antibodies created,” explains Professor Eyer. By separating individual cells and keeping them in a controlled environment, researchers can assess the quantity and quality of a specific antibody produced by a single cell at a given time. “If you have two cells that produce antibodies, then you’re going to measure a mixture of the two, so it’s important for us in the project to keep them separate,” says Professor Eyer.
This approach is now being used in the project to investigate the various different antibodies that can be produced in the body.
Each of the estimated 5,000 or so antibodies in a healthy individual is comprised of different sequences of amino acids, and the nature of that sequence determines what antigen they recognise. “An individual will have a certain set of antibodies against tetanus, let’s say, a certain set of antibodies against diphtheria, and so on. For every antigen you encounter, you produce a repertoire of antibodies,” says Professor Eyer. There are around 100 different sequences of antibodies in the body capable of binding to a specific antigen, which adds up to a highly complex overall picture. “An individual
can generate around 1012 different sequences, or even more, so there’s a huge space that your adaptive immune system can explore,” continues Professor Eyer. “Typically certain solutions are more likely to ‘win’ than others. Therefore, the team focuses their analysis on the functionality, binding strength, specificity and functionality – parameters that cannot (yet) be easily linked to the amino acid sequence.”
Researchers are both measuring the quantity of antibodies produced by a cell and also analysing the quality. There is a high degree of variability in the amount of antibodies that different cells
Lastly, the quantity and quality of antibodies is not stable over an immune response. The immune response is highly dynamic, and there are many different factors to take into account when following these functional repertoires of antibodies over time. Professor Eyer and his colleagues change variables like the vaccine composition or the day of analysis, then assess the condition of the cells, making use of the two points of flexibility of the murine model system.. “In individuals undergoing a second immunisation, we see a lot of reactivation of previously formed cells early
duration of protection for individuals who don’t respond to current vaccines. While vaccines are an important part of health protection, a certain proportion of the population do not respond effectively. “With hepatitis B for example, we see that around 10 percent of people who get vaccinated are not protected from the vaccination. We want to understand how we can use knowledge gained from these assays, this way of quantifying functionality, to design new vaccines for people who do not respond to a standard vaccine,” outlines Professor Eyer. The hope is to eventually move towards a more targeted approach to vaccination, rather than administering the same vaccine to everybody. “For example, individuals with a certain risk profile might benefit from assembly A, while others might benefit from assembly B,” explains Professor Eyer. “This could help us to reduce severe side effects even further, and induce optimal responses for each individual.”
The project’s findings are unlikely to be immediately translated into clinical development, as this research is still at a fairly early stage. However, Professor Eyer has cofounded a spin-off company called Saber Bio SAS building on some of the project’s work, and there is wider interest in using these single-cell assays. “We have also started to work together with clinicians in autoimmune and –inflammatory diseases,” he says. Here, these areas might profit from novel technologies that allow to quantify the quality
of immune responses, albeit in these unwanted field results. In terms of the projects beyond FuncMab, the group works on developing new assays with the ultimate goal of improving the diagnosis and personalised treatment of autoimmune and inflammatory diseases. “We hope to finish our experiments in early 2024, and then we can look to build upon our findings,” he continues. “Open Science is important to use so that other researchers can use our assays, either by themselves, through clinical cooperations, or via Saber Bio. This includes not only looking at antibody producing cells, but a lot of other cells and functionalities as well, which we hope will have a positive impact on the lives of patients and their families.”
Fluorescent image of droplets. The fluorescent line indicates the presence of a functionally active cell, in this case of an antibodysecreting cell in the droplets.
FunCMAB
High-throughput single-cell phenotypic analysis of functional antibody repertoires
Project Objectives
Different threats (viruses, bacteria, toxins) require distinct functional repertoires for effective immune responses. We propose monitoring and quantifying these functions on the monoclonal antibody level to assess vaccine candidates to tailor vaccines for optimal responses. The project aims to develop new analytical approaches for vaccine development and comprehension of vaccine-induced protection.
Project Funding
This project has received funding from the European Union’s Horizon 2020 research and innovation programme under Grant agreement ID: 803363.
Project Partners
• Olivia Tamara Mariann Bucheli
• Kevin Portmann
• Ingibjörg Sigvaldadottir
• Nathan Aymerich
• Alessandro Streuli
• Dr. Magda Rybczynska
Contact Details
Brightifeld image of an individual cell in a 50 pL droplet containing magnetic nanoparticles that form a beadline. This assembly can be then used to measure antibody secretion efficiently (see above).
Project Coordinator, Dr Klaus Eyer
Functional Immunerepertoireanalysis
HCI H433; Vladimir-Prelog-Weg 1-5/10 8093 Zürich, Schweiz T: +41 44 633 74 57 E: klaus.eyer@pharma.ethz.ch W: https://eyergroup.ethz.ch/news-andevents.html
may produce within a given timeframe. “Some cells maybe produce 1-2 antibody molecules a second, while there are cells that can produce between 5,000-6,000 in a second,” outlines Professor Eyer. In terms of quality, Professor Eyer and his colleagues consider how well an antibody binds to the health threat, whether it is a virus, bacteria or toxin. “How well does the antibody bind to, say, Epstein-Barr virus? Does it only bind to Epstein-Barr? Does it neutralize? What you want is a specific response that produces the best exchange binding with functionality possible against your antigen that was used for vaccination,” he says. “We look at both the strength of the binding and also the concentration of the antibodies. If you have a lot of weakly-binding antibodies, they can still be very efficient in terms of helping to eliminate an antigen.”
in the immune response. These cells then go back into the secondary immune response,” he says. Building on this work, researchers can then make predictions on how these cells will function in future. “We can see where the cell is, and by following its trajectory, we can identify which cells are most likely to survive and which will probably die in the next 2-3 weeks. We can effectively monitor their health, and also therefore aim to predict the duration of protection,” continues Professor Eyer.
Vaccine variability
This research represents an important contribution to the wider goal of understanding why vaccines work effectively in some people and not others, which is a step towards improving how efficient functional antibody responses are generated, and what defines the
Professor Klaus Eyer is an assistant professor in the Department of Chemistry and Applied Biosciences at ETH Zurich where he leads a group developing and applying novel analytical strategies for the direct, quantitative, and deep-phenotypic dynamic analysis of individual cellular functions. He holds a Doctorate in BioAnalytical Analysis.
We know that antibodies are produced by cells, and that a healthy human being has around 5,000 different antibodies against various diseases in their bloodstream. We want to analyse every antibody, by itself, for its functionality.
single cells: Microscopic image showing water-in-oil droplets (5x10-11 L) either empty (blue dots) or containing a cell (yellow, green dots). The ability to isolate one cell allows us to study the functions of individual cells. The project has developed an approach that measures cellular functionality with single-cell resolution, called ‘DropMap’ (coming from the ‘mapping of droplets’). It is used to analyze immune cell functionalities and study research questions surrounding vaccines and personalized medicine. Photo: Prof. Eyer Klaus (2017), Eyer Group, IPW.