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A Pseudovirus‐Based Method for the Simultaneous Quantitation of Neutralization Antibodies Against All Three Poliovirus Serotypes

Year: 2025

Type: article

Abstract: ABSTRACT Poliovirus is characterized by three antigenically distinct serotypes that do not elicit cross‐neutralizing antibodies. In the final stages of poliovirus eradication, the gold‐standard conventional neutralization test (cNT) for detecting serum neutralizing antibodies (NAbs) is highly restricted due to biosafety concerns. To address this, we developed a high‐throughput, tri‐color pseudovirus‐based neutralization assay (PBNA) for the simultaneous quantification of NAbs against all three poliovirus serotypes. We generated pseudoviruses by co‐transfecting cells with P1 plasmids, a replication plasmid, and a T7 RNA polymerase plasmid. By optimizing P1 expression, sensitive cell selection (HEK 293T), and plasmid transfection ratios (3:3:1 for P1, replicon, and T7 plasmids), we produced high‐titer pseudoviruses (>29‐fold increase in titers). Based on high‐titer pseudovirus encoding distinct fluorophores (E2, eGFP, and RFP), the PBNA was established, which was optimized for a 12 h incubation period, 4 × 10⁴ cells per well, and 1500 TCID 50 /mL of pseudovirus. It demonstrated high sensitivity, strong serotype specificity, and excellent reproducibility. Furthermore, the PBNA and cNT exhibited excellent congruency ( r > 0.88, all serotypes). The tri‐color PBNA provides a safe, rapid, and alternative to the cNT, making it an invaluable tool for large‐scale serosurveillance, novel vaccine evaluation, and fundamental virological investigations in the post‐eradication era.

Source: MedComm

DOI: 10.1002/mco2.70551