Polyomavirus JC (JCV) is the causative agent of progressive multifocal leukoencephalopathy (PML), a rare and frequently fatal brain disease that afflicts a small fraction of the immune-compromised population, including those affected by AIDS and transplantation recipients on immunosuppressive drug therapy. Currently there is no specific therapy for PML. The major capsid viral protein 1 (VP1) involved in binding to sialic acid cell receptors is believed to be a key player in pathogenesis. PML-specific mutations in JCV VP1 sequences present at the binding pocket of sialic acid cell receptors, such as L55F and S269F, abolish sialic acid recognition and might favor PML onset. Early diagnosis of these PML-specific mutations may help identify patients at high risk of PML, thus reducing the risks associated with immunosuppressive therapy. As a first step in the development of such early diagnostic tools, we report identification and characterization of affinity reagents that specifically recognize PML-specific mutations in VP1 variants using phage display technology. We first identified 2 peptides targeting wild type VP1 with moderate specificity. Fine-tuning via selection of biased libraries designed based on 2 parental peptides yielded peptides with different, yet still moderate, bindinspecificities. In contrast, we had great success in identifying synthetic antibodies that recognize one of the PML-specific mutations (L55F) with high specificity from the phage-displayed libraries. These peptides and synthetic antibodies represent potential candidates for developing tailored immune-based assays for PML risk stratification in addition to complementing affinity reagents currently available for the study of PML and JCV.
Keywords: BSA, bovine serum albumin; CDR, complementarity determining region; CSF, cerebrospinal fluid; D66H, Asp to His mutation at position 66; DHFR, dihydrofolate reductase; ELISA, enzyme linked immunosorbent assay; HRP, horseradish peroxidase; IPTG, isopropyl β-D-1-thiogalactopyranoside; JC virus; JCV, polyomavirus JC; L55F, Leu to Phe mutation at position 55; P8, M13 major coat protein; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PML, progressive multifocal leukoencephalopathy; S269F, Ser to Phe mutation at position 269; TMB, 3,3',5,5'-tetramethylbenzidine; VLP, virus-like particle; VP1, major capsid viral protein 1; WT: type 3 wild type JCV VP1; phage display; protein engineering; synthetic antibody; virus-like particle.