Development and Evaluation of a Baculovirus-Expressed VP1-Based Immunoassay for BK Polyomavirus Serology and Preliminary Seroepidemiological Applications.

Abstract

BK polyomavirus (BKPyV) is highly prevalent and clinically relevant in immunocompromised populations, underscoring the need for reliable serological tools to support epidemiological monitoring and clinical follow-up. This study aimed to generate a recombinant VP1-based enzyme-linked immunosorbent assay (ELISA) using a baculovirus-Sf9 insect cell system and to explore its application in a human serum cohort. Recombinant VP1 was produced by baculovirus-mediated expression, purified by nickel-nitrilotriacetic acid affinity chromatography, and used to establish an indirect IgG ELISA. A plasmid-based transient expression approach in Sf9 cells was evaluated only as a feasibility experiment and did not yield sufficient VP1 for assay development. Analytical performance was assessed using a polymerase chain reaction (PCR)-defined serum panel (n = 30), and the assay was subsequently applied to 149 sera with unknown BKPyV status. Baculovirus-mediated expression generated high-purity VP1 with an average yield of approximately 1.55 mg/mL from a 10 mL culture. PCR-confirmed positive sera showed markedly higher optical density values than PCR-negative sera, with very good analytical discrimination in this small validation set (area under the curve = 0.996). In the cohort analysis, overall seroreactivity was 89.7%, antibody reactivity increased with age, and higher ELISA signals were observed in renal transplant and hemodialysis patients than in healthy individuals. Detectable reactivity in young children may reflect early exposure or passive maternal antibodies. These findings indicate that insect cell-derived BKPyV VP1 is a suitable antigen for serological assays and a practical platform for preliminary seroepidemiological applications, which require confirmation in larger independent validation cohorts.