Background/Purpose:

An unbiased and comprehensive approach to the analysis of Sjögren’s syndrome (SS) antibody repertoires would provide important data on its pathogenesis. Phage ImmunoPrecipitation sequencing (PhIP-seq) utilizes next generation DNA sequencing analysis of a phage-displayed human peptidome to characterize the binding specificities of autoantibody repertoires. We utilized PhIP-seq to define the serum autoantibody repertoire in SS, with the goal of revealing novel, shared SS-specific, self-antigens, which are involved in disease pathogenesis.

Methods:

The study cohort consisted of 193 SS patients consecutively evaluated in the Hopkins Sjögren’s Center, each with independent anti-Ro60 [immunoprecipitation (IP) using IVTT generated S³⁵-Ro60] and anti-Ro52/-SSB (ELISA) antibody testing and positive results in 181 (94%). Control cohorts included 47 SLE patients with positive anti-SSA/SSB serology but without SS (Hopkins Lupus Center) and 301 healthy individuals (United Kingdom repository). Serum from each individual was screened against an overlapping 90-mer human peptidome library using automated PhIP-seq. Peptide enrichments were quantified as number of standard deviations from expected abundance, based on analysis of negative control (no serum) IPs (‘z-scores’). For each protein, analyses of the most enriched peptide were used to identify autoantibodies that associate non-randomly among these subsets of SS patients and controls.

Results:

The top SS-associated antibodies were anti-Ro60 and anti-Ro52, the benchmark SS autoantibodies. When analyzed using a z-score cutoff of 10 and compared to conventional assay results, the sensitivity and specificity of PhIP-seq autoantibody detection in the SS cohort was 49% and 88% for anti-Ro60, 84% and 88% for anti-Ro52, and 32% and 99% for anti-SSB. By PhIP-seq, most anti-SSA positive SS patients had either anti-Ro52 alone (40.6%) or both anti-Ro52 and -Ro60 (58.6%), but rarely anti-Ro60 alone (0.8%). In both SS and SLE controls, anti-Ro52 was directed primarily to the 1-90 and 136-205 amino acid epitopes and anti-Ro60 to the 181-270 and 226-295 amino acid epitopes. Using a minimum frequency cut-off of 3% and a SS-association of 3-fold versus lupus and healthy controls, we identified antibodies to 4 autoantigens, including 2 previously discovered SS-associated minor antigens.

Conclusion:

A comprehensive analysis of the antibody repertoire to the complete human peptidome in SS using PhIP-seq did not reveal a predominant novel autoantigen. This technology is limited to detection of antibodies that recognize linear epitopes, and thus may fail to reveal antibodies directed against conformational or post-translationally modified epitopes. In this SS cohort, antibodies to linear Ro60 epitopes were rarely formed in the absence of antibodies targeting linear Ro52 epitopes. The two novel SS-associated autoantigens are undergoing additional validation.

Supported by the Sjogren’s Syndrome and Jerome L. Greene Foundations.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/analysis-of-autoantibody-reactivity-to-the-complete-human-peptidome-by-phage-immunoprecipitation-sequencing-does-not-identify-a-predominant-novel-autoantibody-in-sjogrens-syndrome/