Four-Color Pseudovirus-Based Neutralization Assay: A Rapid Method for Evaluating Neutralizing Antibodies Against Quadrivalent Hand, Foot, and Mouth Disease Vaccine

Figure 1. Selection of four-color fluorescent reporter genes and packaging strategies for pseudoviruses. (A) Four distinct fluorescent protein expression signals can be detected using the instrumentation. (B) Four enterovirus capsid expression plasmids were constructed using a consistent strategy. (C) Strategy for constructing the EV71 replicon. (D) Strategy for constructing the CA16 replicon. (E) Strategy for constructing the CA6 replicon. (F) Strategy for constructing the CA10 replicon. (G) After infecting RD cells with the four-color fluorescent pseudoviruses, excitation/emission light channels at specific wavelengths can be specifically detected. CMV: Cytomegalovirus promoter element; codon-optimized P1: codon-optimized P1 gene; SV40: SV40 polyA signal sequence; pEV71-optiP1, pCA16-optiP1, pCA10-optiP1, and pCA6-optiP1: plasmids encoding the capsids of EV71, CA16, CA10, and CA6, respectively; BFP-2A: 3′-end of BFP fluorescent protein gene linked to 2A peptide (AITTL); GFP-2A: 3′-end of GFP fluorescent protein gene linked to 2A peptide (KITTL); RFP-2A: 3′-end of RFP fluorescent protein gene linked to 2A peptide (KITTL); CFP-2A: 3′-end of CFP fluorescent protein gene link to 2A peptide…

Figure 2. Optimization of four parameters for the pseudovirus-based neutralization assay (PBNA), the specificity as part of method validation, and a correlation analysis of PBNA and cell-based neutralization assay (CBNA) results. (A) Cell line selection for neutralization. Negative results were designated as 0.1; (B) testing time point; (C) pseudovirus addition volume; (D) cell inoculation density. (E) A total of 56 serum samples from nonimmunized mice served as negative controls to establish the cut-off value using four-color PBNA. Cut-off value = Mean + 3SD. (F) Mice or rats were immunized with vaccines against EV71, CA16, CA10, CA6, poliovirus, and HAV to obtain the corresponding antisera. The specificity of the pseudovirus detection results was evaluated using four-color PBNA. (G) The logarithmic conversion of NT50 values and sample dilutions exhibits excellent linearity, with R2 > 0.98. (H) Correlation between PBNA and CBNA test results. CBNA: NT50 was defined as the highest dilution capable of inhibiting 50% of the cytopathic effect. PBNA: NT50 was defined as the reciprocal of the dilution factor that achieved 50% infection inhibition rate. ****: p < 0.0001; ***: p < 0.001; **: p <…