Date

12-16-2025

Department

School of Health Sciences

Degree

Doctor of Philosophy in Health Sciences (PhD)

Chair

Joseph W. Francis

Keywords

Yeast Surface Display, Antibody Discovery, Junín Virus, Fluorescence Activated Cell Sorting

Disciplines

Biochemistry, Biophysics, and Structural Biology | Biomedical Engineering and Bioengineering

Abstract

The importance of this study is to perform antibody discovery as a means to derive novel preventative and therapeutic antibody countermeasures against the Junín virus, to alleviate the potential risk of a global pandemic. The purpose of this methodological study was to develop a protocol that utilizes yeast surface display to discover antibodies, from naïve human antibody libraries, that can bind the Junín Virus antigen. The methodology of yeast surface display employs the galactose-inducible GAL1 promoter to synthesize and express antibody proteins bound to the Aga2p and Aga1p and anchored to the cell wall by glycosyl phosphatidylinositol motif (GPI). The techniques of Fluorescence Activated Cell Sorting and Flow Cytometry provide fluorescence data that corresponds to the levels of antibody protein expression on the surface of the yeast cell and the antibody’s capabilities to bind the Junín virus antigen at the conserved epitopes. This study utilizes quantitative experimental theory to manipulate the variables of binding and expression from antibodies that undergo affinity maturation through the use of Fluorescence Activated Cell Sorting. The data collected demonstrated affinity maturation of the sorted antibodies toward an increase in binding and expression levels. Next Generation Sequencing from Oxford Nanopore provided the capabilities to sequence the long reads of the antibody sequences discovered through Fluorescence Activated Cell Sorting and Yeast Surface Display. Nanopore sequencing of the variable heavy and variable light chain pairs sequenced from the three rounds of Fluorescence Activated Cell Sorting resulted in the discovery of thirty unique antibodies capable of binding the Junín Virus Antigen.

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