Multiplexed, High-Throughput Serum Assays Using Lentivirus Display to Analyze Antibody Responses
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hornick-ahornick-phd-hst-2026-thesis.pdf
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66.86 MB
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3193629526a49884c8e79ac2ca0e7a9e
Author(s)
Hornick, Amanda Claire
Advisor(s)
Birnbaum, Michael E.
Date Issued
February 2026
Publisher
Massachusetts Institute of Technology
Abstract
Antibodies are an integral part of our immune system. Whether generated through antigen exposure or provided as therapeutics, antibodies can direct targeted responses against specific antigens on foreign substances, pathogens, and cells. Therefore, accurately, efficiently, and comprehensively characterizing the antigen recognition capabilities of individuals’ antibody repertoires is critical to understanding individuals’ extent of protection against disease. As existing antibody measurement techniques are limited in their ability to test antibodies’ recognition of multiple, native-conformation antigens in a single reaction, we developed assays that employ libraries of hybrid pseudotyped, genetically barcoded lentiviruses to measure antibodies in a multiplexed, high-throughput manner. To record interactions between library lentiviruses and serum antibodies, our assays utilize cells expressing either an Fc receptor or viral entry receptor to measure antibodies’ binding or neutralization of their target antigens, respectively. During the COVID-19 pandemic, we constructed a lentivirus library with members displaying SARS-CoV-2 spike protein variants and other viral surface proteins to study the effects of infection, vaccination, and monoclonal antibody therapies on antiviral antibody responses. Here, we first discuss the strategies we utilized to expand our lentivirus library as well as optimize and understand the mechanisms underlying our assays. Using sera from pre-pandemic, COVID-19-convalescent, and COVID-19-vaccinated donors, we demonstrate the ability of our assays to differentiate between samples with and without antibodies against antigens of interest, and we validate our assays’ readouts against those from established immunoassays. In preparation for evaluating how monoclonal antibody therapies can affect patients’ antiviral antibody responses, we also explore methods by which readout-confounding therapeutic antibodies can be removed from serum samples. Finally, we highlight how our assays could be applied in other disease contexts by generating allergen-displaying lentiviruses. Through additional engineering and optimization of the lentivirus libraries and receptor-expressing cell lines used in our assays, we can further increase the throughput and multiplexability of antibody repertoire profiling and enhance our ability to characterize how different diseases and medical interventions impact the development and maintenance of antigen-specific antibody responses.
MIT Department
Harvard-MIT Program in Health Sciences and Technology
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