Discovery of a Pre-Eclampsia Associated Antibody That Binds to a Viral Antigen and Human Protein

Diverse diseases, including autoimmune conditions such as celiac disease and myasthenia gravis [1], have been proposed to involve environmental factors.  While the immune system archives the response to environmental exposures in the form of an antibody repertoire, it has been difficult to impartially identify which molecules from this repertoire are associated with disease. To address this issue, we screened bacterial-displayed peptide libraries to identify peptides with enhanced binding to antibodies present in a diseased state over normally present antibodies and characterized the disease-associated binding motifs.  We applied this quantitative discovery process to identify molecular diagnostics for pre-eclampsia (PE), a condition with unknown etiology that affects 5-8% of pregnancies.  Previously characterized autoantibodies that bind the angiotensin II type 1 receptor in PE patients [2][3] are difficult to detect, vary in prevalence amongst studies, and most importantly, lack specificity. Thus, we hypothesized that additional PE-specific antibody biomarkers may exist. 

Screening a fully randomized peptide library against pools of diluted plasma from PE and healthy outcome pregnancies (HOP) reproducibly identified a peptide binding motif with strong similarity to a region of a common viral antigen (VA), and we used directed evolution to expand this motif.  Additionally, by combining peptide library screening with next-generation sequencing, we profiled antibody binding specificities of individuals and confirmed enrichment of this viral antigen linked motif amongst PE patients.  Importantly, we determined that antibody binding to a bacterial-displayed 15-mer fragment of the VA containing this motif (VA15) correctly distinguished 67% of PE patients (n=42) and 86% of HOP (n = 43) at or near term.  Furthermore, we linked this motif and verified antibody binding activity to a fragment of a human G protein-coupled receptor (hGPCRa), while the synthetic VA15 competed with this antibody binding.  Moreover, VA15 inhibited antibody binding to the full-length hGPCRa overexpressed in HEK293T cells and natively expressed in a model trophoblast cell line. Finally, we verified the presence of hGPCRa in PE placental tissue. 

Our results demonstrate that an unbiased quantitative molecular separation process identifies disease-specific antibody binding peptide motifs.  Furthermore, these binding motifs can be used to identify environmental antigens and human protein targets of antibodies associated with a disease.  Thus, these antibody detecting peptides can serve as novel molecular diagnostic tools and help elucidate potential therapeutic targets.

[1] Cusick, M., Libbey, J. and Fujinami, R. Molecular Mimicry as a Mechanism of Autoimmune Disease. Clinical Reviews in Allergy and Immunology 42,102–111 (2012).

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

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