(6f) Discovery of Antibody Biomarkers Present in Pre-Eclampsia

Discovery of biologic molecules specific to a diseased state, or biomarkers, can lead to diagnostic development, therapeutic target identification, and improved understanding of disease pathogenesis.  Antibodies remain an attractive class of biomarkers given their amplification by the immune system, stability, and current clinical use.  In this work, we demonstrate the utility of bacteria-displayed peptide libraries in conjunction with fluorescence activated cell sorting (FACS) to identify the presence of novel antibody biomarkers by quantitatively screening for peptides with enhanced binding to antibodies present in a diseased state over normally present antibodies. We applied these methods in an effort to identify molecular diagnostics for pre-eclampsia (PE), a condition with unknown etiology that affects 5-8% of pregnancies.  Previous studies have implicated a pathologic role for autoantibodies against a specific epitope on the angiotensin II AT₁ receptor in PE patients [1][2].  However, these antibodies remain difficult to detect, vary in prevalence amongst studies, and most importantly, lack specificity.  We hypothesized that additional PE specific antibody biomarkers may exist, and this complex disease could serve as a model for developing methodologies to identify novel antibody biomarkers using the known epitope as a control.  Therefore, we expressed the seven amino acid epitope on the surface of E. coli to determine the cross-reactivity and specificity in a set of 45 PE and 48 normal-outcome pregnancies (N), and we applied two distinct screening strategies to a fully randomized 15 amino acid bacteria-displayed peptide library with a subset of this cohort.  At an optimized cutoff, the cell-surface expressed AT₁ epitope achieved 78% sensitivity (35/45 PE) and 56% specificity (27/48 N).  For screening, we used either antibody enriched fractions or unprocessed, diluted plasma.  Although both screens resulted in peptides with significantly (p < 0.05) higher PE reactivity than N and diagnostic potential in a validation set, sequence analysis yielded a stronger consensus family with three subgroups from the dilute plasma screen.  Based upon this consensus, we created and screened a focused library to further evolve the motif and enhance sensitivity. This work demonstrates the utility of bacteria-displayed libraries to identify novel diagnostic reagents for antibody biomarker detection in PE.  Furthermore, the improved consensus motif may enable identification of antigens mimicked by these antibody binding peptides. The present methodology may thus improve understanding of disease pathogenesis and guide therapeutic development.    

[1]  Wallukat, G. et al. Patients with Preeclampsia Develop Agonistic Autoantibodies against the Angiotensin AT₁ Receptor. The Journal of Clinical Investigation 103,945–952 (1999).

[2] Zhou, C. C. et al. Angiotensin Receptor Agonistic Autoantibodies Induce Pre-eclampsia in Pregnant Mice. Nature Medicine 14, 855–862 (2008).