Development of Methods that Mimic, Measure, and Detect Evasion of the Adaptive Immune System
CitationFujimura, Eric. 2021. Development of Methods that Mimic, Measure, and Detect Evasion of the Adaptive Immune System. Doctoral dissertation, Harvard University Graduate School of Arts and Sciences.
AbstractThe ability of the adaptive immune system to generate incredible genetic diversity in response to environmental stimuli provides both a unique challenge to develop methods that can comprehensively analyze immune repertoires, and an inspiration for novel technologies that attempt to mimic this capability for engineering applications. I am working on a number of technology development projects that encompass both faces of this intersection between synthetic biology and immunology. The first technology I have developed is a system for targeted in vivo mutagenesis using transcriptionally directed DNA deamination. This technology was designed to mimic the in vivo method used to enhance the functionality of antibody genes. The technology combines phage RNA polymerases and base editors that cause nucleotide deamination and mutagenesis of target genes throughout their coding regions. The second technology I have used is antibody detection methods that allow deep saturation analysis of antibody binding sites on the SARS-Co V-2 pathogen. Deep serological profiling of 232 COVID-19 patients and 190 pre-COVID-19 era controls using VirScan revealed over 800 epitopes in the SARS-CoV-2 proteome, including 10 epitopes likely recognized by neutralizing antibodies A machine learning model trained on VirScan data predicted SARS-Co V-2 exposure history with 99% sensitivity and 98% specificity; a rapid Luminex-based diagnostic was developed from the most discriminatory SARS-Co V-2 peptides. Individuals with more severe COVID-19 exhibited stronger and broader SARS-Co V-2 responses, weaker antibody responses to prior infections, and higher incidence of CMV and HSV-1, possibly influenced by demographic covariates. Among hospitalized patients, males make greater SARS-Co V-2 antibody responses than females. Lastly, I developed a synthetic virome-wide ORF collection ribosome display-based methodology for pan-viral serological profiling. This library was applied in forward genetic screens to study viral mechanisms of escape from adaptive immunity in three different branches: regulation of antigen presentation, inhibition of IFN-y signaling and evasion of T-cell killing. Genetic screening of antigen presentation using this virome wide ORF collection revealed several canonical and novel viral regulators of MHC-I cell surface expression. Among novel suppressors of cell surface MHC-I was MC162R, a gene encoded by the Poxvirus Molluscum Contagiosum Virus (MCV). In depth mechanistic analysis revealed that MC162R acts through dislocation of cell surface MHC and colocalization with MHC in intracellular vesicles. Genetic suppressor screening revealed that an E3 ligase ITCH and components of the ESCR T-0 complex are necessary for downregulation of cell surface MHC in MC162R expressing cells. Downregulation of MHC was further dependent on the PY motifs in MC 162R protein as well as the lysosome. These observations lead us to the model where MC162R interaction with MHC induces its delivery to the lysosome through ubiquitination by ITCH and the ESCRT-0 complex.
Citable link to this pagehttps://nrs.harvard.edu/URN-3:HUL.INSTREPOS:37369987
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