Engineering the fusion machinery core of SARS-CoV-2 spike protein for vaccine immunogen design

The emergence and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in 2019 prompted the rapid development and deployment of vaccines harboring the spike protein of the virus. For vaccines to be effective, the spike protein immunogen required engineering into its prefusion-stabilizing conformation to elicit protective antibodies that primarily bind the spike receptor-binding domain (RBD), in addition to the spike’s N-terminal domain, and fusion machinery core. I showed that natural SARS-CoV-2 infection or vaccination with SARS-CoV-2 spike protein elicits protective, public antibodies encoded by IGHV3-53/3-66 with unique amino acid sequence signatures. Eliciting these public RBD antibodies is an alluring goal of vaccination for prophylactic protection among majority of the human population. To this end, I developed a systematic and unbiased high-throughput method of identifying prefusion-stabilizing mutations in the fusion machinery core of SARS-CoV-2 spike protein that will render the RBD intact. By combining saturation mutagenesis, mammalian cell display, a cell-based fusion assay, fluorescence-activated cell sorting, and deep sequencing, I identified several mutations, in addition to the two proline mutations used in currently approved vaccines, that lock the spike protein in its prefusion conformation. Biophysical characterization revealed that a D994Q mutation in the first heptad repeat prevented the complete unfolding of the spike trimer by forming an additional intra-protomer hydrogen bond. To develop vaccines that can target future variants, escape mutants of the spike protein need to be identified and characterized. Public antibodies encoded by IGHV1-69 and IGKV3-11 target the highly conserved S2 subunit containing the fusion machinery core of SARS-CoV-2. However, deep mutational scanning experiments showed that the D950N and Q954H mutations found in Delta and Omicron variants, respectively, weaken the binding of public antibodies to spike, and consequently, decrease protection in vivo. These results highlight that an innovative, high-throughput, deep mutational scanning-based method can accelerate vaccine immunogen engineering by systematically interrogating each mutation, and antigenic drift of immunogenic proteins provide a significant challenge for the proactive development of universal vaccines.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

Dissertation

Discipline thesis:degree_discipline

Biophysics & Quant Biology

Grantor

University of Illinois Urbana-Champaign

Year dc:date

2025

Author dc:creator

• Tan, Timothy James

Contributors dc:contributor

• Wu, Nicholas C
• Brooke, Christopher B
• Stadtmueller, Beth M
• van der Donk, Wilfred A

• Vaccine design
• Virology
• Antibodies
• Protein engineering

Statement dc:rights

• Copyright 2025 Timothy James Tan

Language dc:language

en, eng

Handle dc:identifier

https://hdl.handle.net/2142/129363

OAI record oai:identifier

https://www.ideals.illinois.edu/oai-pmh?verb=GetRecord&metadataPrefix=etdms&identifier=oai%3Awww.ideals.illinois.edu%3A2142%2F129363