Iterative immunoprecipitation and phage pre-wash dramatically improve epitope-resolved serology by VirScan

Area of Science:

• Immunology
• Virology
• Systems Biology

Background:

• Accurate mapping of antibody epitope repertoires is crucial for understanding immune responses to infections and vaccinations.
• Phage immunoprecipitation sequencing (PhIP-seq), including VirScan, provides single-peptide resolution but faces limitations like low signal-to-noise ratio and non-specific binding.
• Distinguishing true antibody-antigen interactions from background noise is essential for reliable serological measurements.

Purpose of the Study:

• To systematically evaluate experimental variables impacting PhIP-seq performance.
• To identify modifications that enhance epitope-level viral serology.
• To develop a more accurate and quantitative workflow for mapping viral antibody repertoires.

Main Methods:

• Systematic evaluation of key experimental variables in PhIP-seq.
• Implementation of two synergistic modifications: iterative immunoprecipitation and serum pre-washing with wild-type phage.
• Validation across healthy donor, HIV-seropositive, and influenza-vaccinated rabbit sera.

Main Results:

• The optimized workflow significantly expanded the enriched peptide population and increased normalized peptide counts.
• Improved separation of viral epitopes from background noise was achieved while maintaining global library representation.
• Quantitative detection of HIV epitopes and identification of strain-specific influenza epitopes were demonstrated.

Conclusions:

• The optimized PhIP-seq workflow provides a reproducible and generalizable method for enhancing viral epitope discovery and serosurveillance.
• This enhanced framework improves the accuracy and quantitative inference of viral antibody repertoires, strengthening VirScan's utility in systems virology.
• The protocol enables more sensitive mapping of vaccine responses and immune histories.