Charged scanning mutagenesis as a high-throughput approach for epitope mapping.

Area of Science:

• Virology
• Immunology
• Biochemistry

Background:

• Identifying neutralizing epitopes is crucial for developing effective vaccines and antiviral inhibitors.
• Charged residue substitutions (e.g., Aspartic acid, Arginine) are well-tolerated at exposed antigen positions and minimally impact protein stability.
• Such substitutions at epitope residues can significantly disrupt binding to cognate partners.

Purpose of the Study:

• To develop and validate a rapid method for epitope mapping using barcoded charged scanning mutagenesis.
• To identify neutralizing epitopes targeted by polyclonal sera against SARS-CoV-2 immunogens.
• To accelerate the development of vaccines and inhibitors by improving interaction site identification.

Main Methods:

• Yeast surface display of barcoded charged scanning mutagenesis libraries.
• Screening using flow cytometry combined with deep sequencing.
• Construction of an Aspartate scanning library for the SARS-CoV-2 receptor binding domain, with each mutation linked to a unique barcode.

Main Results:

• The developed method successfully mapped epitopes targeted in mice immunized with SARS-CoV-2 immunogens.
• Charged scanning mutagenesis with barcoding utilizes libraries with >50-fold lower diversity compared to complete mutational scans.
• The approach facilitates library construction, screening, downstream analysis, and multiplexing of samples.

Conclusions:

• This barcoded charged scanning mutagenesis approach offers a faster and more efficient method for epitope mapping.
• The technique accelerates the identification of critical interaction sites on viral antigens.
• This advancement holds significant potential for expediting vaccine and inhibitor development against viral pathogens.