Introduction:: The adaptive immune response provides crucial protection against pathogens including viruses. Antibodies produced by B cells are central to this immune response, both inhibiting viral replication and driving immune effector functions. Despite the importance of antibodies in viral immunity and vaccine development, dissecting the factors that contribute to the selection of potent and protective antibodies remains a challenge. We hypothesize that the accessibility of epitopes presented on the viral surface is one such factor, influencing B cell engagement and activation. However, testing this hypothesis has traditionally been difficult: epitopes that vary in their accessibility typically also vary in their sequence and structure, making it difficult to decouple the contributions of these factors in driving B cell selection.
Materials and Methods:: We have designed an experimental system using genetically engineered human B cells to understand the role of epitope accessibility in BCR engagement and B cell activation. We developed these engineered B cells by knocking-out the endogenous B cell receptor (BCR) and knocking-in single-chain BCRs that express tags for enrichment and fluorescent labeling (Figure 1A). These engineered BCRs are derived from antibodies that target epitopes on the fusion protein of respiratory syncytial virus (RSV). Importantly, these epitopes can be presented in either membrane-proximal or membrane-distal conformations that differ in their accessibility (Figure 1B). Using fluorescence microscopy, we track the activation and engagement of these RSV-specific B cells when they are presented with viral antigens reversibly tethered to coverslips via Erythrina cristagalli lectin (ECL). To determine specificity of the engineered B cells and quantify epitope accessibility, we have developed epitope masking experiments, where we titrate soluble antibodies that will block the epitope that the B cell recognizes. By titrating the concentration of competing antibody and measuring the efficiency of B cell antigen extraction, we can determine the sensitivity of a particular BCR to epitope masking.
Results, Conclusions, and Discussions:: For multiple engineered RSV-specific B cells, we observed extraction of RSV particles bound to ECL-coated coverslips using fluorescence timelapse microscopy. We observed enrichment and colocalization of the BCR around the RSV particles, indicating specific BCR engagement with viral antigen (Figure 1C). This engagement was inhibited by competing antibodies, demonstrating epitope specificity of the engineered B cells. These results establish the use of engineered B cells with well-defined BCR specificity to understand how the conformation of RSV F influences epitope accessibility and BCR engagement. Experiments comparing BCR engagement and antigen uptake across multiple RSV-specific B cells where epitope accessibility varies are currently in progress. This project will provide new insights into the host immune response to viral infections and immunodominance hierarchies that have been observed for RSV and other human viruses.
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This work is supported by NIH Grant #1R21AI163985 and NSF Grant # 2238165