(601a) An Antibody Associated with Pre-Eclampsia Binds to an Environmental 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 developed a molecular discovery process to identify the targets of antibodies in patients with disease. This method uses bacterial-displayed peptide libraries in conjunction with fluorescence activated cell sorting (FACS) to quantitatively screen for peptides with enhanced binding to antibodies present in a diseased state over normally present antibodies. We applied this 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. 

Applying this quantitative molecular separation process, we previously identified a peptide binding motif with strong similarity to a region of a common viral antigen (VA) and used directed evolution to expand this motif.  Importantly, we validated that antibody binding of a 15-mer fragment containing this motif from VA correctly distinguished 65% of PE patients (n=43) and 83% of healthy outcome pregnancies (n = 47) 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).  Thus, we hypothesized that molecular mimicry may be involved in PE.  We tested three pools of PE patients’ plasma and demonstrated that the synthetic 15-mer from VA competed with antibody binding to the bacterial-displayed hGPCRa fragment.  Moreover, the VA peptide inhibited antibody binding to the full-length hGPCRa over expressed in HEK293T cells.  Additionally, we assessed whether these VA/hGPCRa binding antibodies demonstrated activity against hGPCRa in human placental tissue.  We next sought to determine the predictive utility of these PE-specific antibody detecting peptides.  Therefore, we evaluated antibody binding activity to the bacterial-displayed VA and hGPCRa fragments using samples taken during the first trimester from healthy women and those who later developed PE. 

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.F., Libbey, J.E. and Fujinami, R.S. 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).