A simple and safe antibody neutralization assay based on polio pseudoviruses

Figure 1.. Schematic view of PV capsid expression vector, replicon RNA， and the production of pseudovirus in 293FT cells. 293FT cells were first transfected with the pcDNA6.0-P1-EGFP plasmid to express P1 proteins, followed by transfection with the replicon RNA. As an encapsulated gene fragment, replicon RNA serves as a template for the synthesis of P2 and P3 proteins, whereas the 2Apro protease releases EGFP and luciferase and cleaves P1 into VP1-VP4 capsid proteins to facilitate pseudoviral packaging.

Figure 2.. Characterization of pseudo-PVs. (a) Expression of pseudovirus capsid protein P1 and fluorescent protein EGFP fusion. 293FT cells transfected with capsid protein vectors produced green fluorescence, indicating successful expression of the proteins. (b) Western blot analysis for the verification of the expression of pseudovirus capsid protein VP1. Lane 1 is a positive control with monovalent sIPV vaccine corresponding to each pseudovirus type; lane 2 represents concentrated samples collected from Mahoney, MEF-1, and Saukett pseudovirus types; lane 3 is a negative control with supernatant harvested from 293FT cells. The antibodies used in the assay were monovalent rabbit antibodies against the virus and HRP-conjugated mouse anti-rabbit IgG. All three pseudoviruses and positive control show VP1 band. (c) Visualization of Mahoney, MEF-1, and Saukett pseudovirions using transmission electron microscopy: diameters are approximately 30nm, with the morphology similar to the wt virus (pointed by in-figure arrowheads). (d) Neutralization curves of PV pseudovirus with mouse anti-PV sera of three different types showed the type-specificity for the neutralization of pseudovirus (Each type…

Figure 3.. Optimization of the pNT method. (a) Linear relationship between dilution and luciferase activity. A linear relationship was observed with pseudoviral titer ranging from 50 to 1,600 CCID50/50 µL. Mahoney R2 = 0.996, MEF-1 R2 = 0.997, and Saukett R2 = 0.999. It is of note that the titer in the x-axis has been converted into lg form. Each dilution depicts average values from eight wells (replicates). Given the cell growth condition, 100 CCID50/50 µL was chosen as the challenge amounts of pseudovirus in pNT. (b) RLU-time curves of 100CCID50 pseudo-PV expressing luciferase. Increasing intensity of the fluorescent signal was observed over time, with a plateau being observed after 8h for pseudo-Mahoney and pseudo-MEF-1 respectively, while it took 14h for pseudo-Saukett to reach the plateau, with each time point depicting average values from eight wells (replicates). Therefore, 12h was chosen for the final RLU readout for all three serotypes.

Figure 4.. The agreement analysis between pNT and cNT. The pNT results of 120 post-vaccination serum samples from phase II clinical trials of sIPV vaccines were compared with those generated with cNT in which wt viruses were used. Spearman correlation statistical analysis showed it is statistically significant between pNT and cNT (p < 0.0001) for three serotypes respectively, with coefficients r = 0.8989 (0.8563, 0.9294) for Mahoney, r = 0.8533 (0.7938, 0.8966) for MEF-1 and r = 0.8013 (0.7238, 0.8588) for Saukett.