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High-throughput single-cell phenotypic analysis of functional antibody repertoires

Periodic Reporting for period 2 - FuncMAB (High-throughput single-cell phenotypic analysis of functional antibody repertoires)

Reporting period: 2019-08-01 to 2021-01-31

Vaccines have had a tremendously positive effect on individual and global health for decades. However, the understanding of their mechanism of action and induced immunological responses is still limited. The concept of vaccine-mediated protection and the quantitation thereof remain especially elusive. Indeed, protection is empirically assayed during clinical trials, and levels and thresholds are defined over time. Here, current testing methods for protection do not provide temporal, spatial, or analytical resolution to expose fundamental differences in the underlying mechanisms that ultimately mediate protection, and therefore the gained understanding is limited. Indeed, immune responses are highly dynamic, heterogeneous and their successful completion involves and needs many different interactions, cells, and functions throughout the organism. Individual cells are the functional units within any immune response, and their varying frequencies and degrees of activity shape and define the response. Therefore, cells from the immune system, their state, activation and ultimately functionality display high dynamic heterogeneity, and there is hence a need for quantitative high-throughput systems that allow for a dynamic, functional single-cell phenotyping, linking function to the individual cells.
Therefore, within this project, we aim to measure, understand, and exploit antibody-mediated vaccine-induced protection on the individual cell and antibody level. By doing so, we aim to not only measure and describe the functional antibody repertoires with single-cell resolution, but also to understand the influences that are introduced by varying vaccination strategies and measure the impact on the quality, quantity, and functionality of the humoral immune response. The overarching objective of this proposal is to quantitatively map antibody functions on the single-cell level, and to use these data sets to understand the selection mechanisms involved in their generation, evolution, and transfer to memory; and to finally exploit the measurement to screen for therapeutic candidates and to accelerate vaccine development.
After building up my research group at ETH, we started developing and integrating several new bioassays to functionally characterize secondary antibody functionalities (neutralization, complement deposition, antibody-dependent cellular cytotoxicity) on a single-antibody level. We have made several substantial contributions to this research project over the last 1.5 years, and towards our general goal of quantifying vaccine-mediated protection. We have established novel ways to quantify the quality of induced humoral repertoires in immunized mice, and identified potential days of interest to study protection (Eyer et al., Journal of Immunology, 2020), have been actively involved in the sharing of our technology (Bounab, Eyer, et al., Nature Protocols, 2020), and in its application in a variety of research questions (Rybczynska et al., Vaccine, 2020, Heo et al., Communications in Biology, 2020, Gérard et al., Nature Biotechnology, 2020). Furthermore, we used our generated data to work in collaboration with theoretical physicist to model the immune response (Molari et al., eLife, 2020), and extracted valuable information about the humoral immune response from this effort. Together with Prof. Oxenius (D-BIOL), Stadler, and Reddy (BSSE), we applied the method to study and describe acute lymphocytic choriomeningitis virus (LCMV) infection in mice (Kräutler et al., Cell Reports, 2020). We are currently finishing up assay development and will start to apply our assays to generate data sets for the advanced goals of this action.
We develop and use a new, cutting-edge technology to shed light into complex immune reactions, with the goal to firstly quantify, secondly understand, and lastly influence the immunological reaction upon vaccination. Our novel bioassays to functionally characterize secondary antibody functionalities are well-beyond the state-of-art and allow us to study the antibody repertoire with unique resolution. The objective of this project is not only to develop new analytical approaches to be able to quantitatively map antibody functions with individual cell resolution, but to use these data sets to understand the underlying selection mechanisms involved in their generation, evolution and transfer to memory of the antibody repertoires. Therefore, the overarching objective of this proposal is to quantitatively map antibody functions on the single-cell level, and to use these data sets to understand the selection mechanisms involved in their generation, evolution, and transfer to memory; and to finally exploit the measurement to screen for therapeutic candidates and to accelerate vaccine development. Multiple variations of these data sets allow separating the influence of different vaccine components, dose and kinetics, as well as the cells from the immune system themselves.
An immobilized droplet array used to measure antibody functions within this action