Selection and Characterization of Novel Glucuronoxylomannan-Mimetic Peptides for Potential Diagnosis of Cryptococcosis

Abstract Cryptococcus neoformans is an encapsulated fungal pathogen characterized by a thick polysaccharide capsule, primarily composed of glucuronoxylomannan (GXM), which represents its major virulence factor and a key target for immunodiagnosis. Mimetic peptides that reproduce GXM epitopes may provide standardized, stable alternatives to native polysaccharide antigens for diagnostic and therapeutic applications. This study aimed to identify and characterize novel GXM-mimetic peptides using phage display technology and evaluate their potential as synthetic reagents for the serological diagnosis of cryptococcosis. Phage display biopanning was performed through three successive cycles using a Ph.D.-7™ combinatorial library against the anti-GXM monoclonal antibody (mAb) 18B7. Selected phage clones were sequenced, and the predominant peptide motif was identified. Based on the consensus sequence, three variants were designed: a linear peptide (LP), a cyclic peptide (CP), and a double-cyclic peptide (DP). Their immunoreactivity and diagnostic performance were evaluated via enzyme-linked immunosorbent assay (ELISA), including dose-response, serum reactivity across clinical cohorts (active cryptococcosis, treated patients, and healthy controls), and competitive inhibition assays against native GXM. Biopanning resulted in significant enrichment, with the peptide PPSLYWL (CryMab 1) emerging as the predominant sequence (54.8% frequency). ELISA demonstrated dose-dependent binding of the synthetic variants to mAb 18B7 and effective discrimination between sera from active cryptococcosis patients and control groups. In competitive ELISA, the LP variant successfully inhibited antibody binding to native GXM by 50.86%, confirming its functional antigenic mimicry. The identified GXM-mimetic peptides, particularly the LP and CP variants, exhibit specific immunoreactivity and diagnostic utility. These findings support their potential as standardized, low-cost alternatives to native fungal antigens in cryptococcosis diagnosis and provide a basis for future peptide-based theranostic strategies.