Mapping of PLA2R Epitopes in Idiopathic Membranous Nephropathy and Clinical Relevance of Epitope Profiles

Background: The M-type phospholipase A2 Receptor (PLA2R) is the main target antigen of idiopathic membranous nephropathy. Four autoantibody-targeted PLA2R epitope domains, including Ricin, CTLD1, CTLD7, and CTLD8, have been identified. Nevertheless, whether the epitope profile is involved in the progression of PLA2R-associated membranous nephropathy remains controversial. Conventional epitope detection techniques have limitations and new methods are needed. Methods: We constructed a yeast surface-displayed PLA2R antigen library by randomly fragmenting the full-length PLA2R gene and expressing them on the yeast surfaces. Fluorescence-activated cell sorting (FACS) was performed with sera from PLA2R-related membranous nephropathy patients. Antigenic epitopes were identified through sequencing and clustering. To analyze large-scale samples in batches, we used representative monoclonal yeasts of each structural domain, obtained through the sorting procedure for flow analysis. Clinical and laboratory data were collected at baseline and follow-up. Associations between epitope numbers and disease conditions and prognosis were analyzed. Results: The capacity, diversity and screening specificity of the PLA2R yeast surface display library were validated. Beyond the reported epitopes, CTLD4, CTLD5, and CTLD6 were newly identified as antigenic targets. In our cohort of 389 idiopathic membranous nephropathy patients, 320 (82%) patients showed epitopes beyond the Ricin domain. The positive rates of CTLD1, CTLD5, and CTLD7 were 53%, 45%, and 54%, while the detection rates of CTLD6, CTLD8, and CTLD4 were lower, at 9%, 8%, and 7%, respectively. The multivariable model demonstrated a 56% higher risk for nephrotic syndrome per epitope increment (OR=1.56, 95%CI 1.28-1.91) after adjustment for gender, age, disease duration, serum creatinine and anti-PLA2R titer. Multivariable Cox regression demonstrated each additional epitope was associated with a 15% lower likelihood in clinical remission (HR 0.85, 95% CI 0.75-0.96), with subsequent mediation analysis revealing anti-PLA2R titer accounted for 12% of this effect. Conclusions: Based on the PLA2R yeast surface display system, we identified three new antigen epitopes of PLA2R in membranous nephropathy. The epitope profiles of PLA2R were associated with disease severity and prognosis of membranous nephropathy.