Development of a Coxsackievirus A16 neutralization assay based on pseudoviruses for measurement of neutralizing antibody titer in human serum

Abstract

Serum neutralizing antibody titers are indicative of protective immunity against Coxsackievirus A16 (CV-A16) and Enterovirus 71 (EV71), the two main etiological agents of hand, foot and mouth disease (HFMD), and provide the basis for evaluating vaccine efficacy. The current CV-A16 neutralization assay based on inhibition of cytopathic effects requires manual microscopic examination, which is time-consuming and labor-intensive. In this study, a high-throughput neutralization assay was developed by employing CV-A16 pseudoviruses expressing luciferase for detecting infectivity in rhabdomyosarcoma (RD) cells and measuring serum viral neutralizing antibodies. Without the need to use infectious CV-A16 strains, the neutralizing antibody titer against CV-A16 could be determined within 15h by measuring luciferase signals by this assay. The pseudovirus CV-A16 neutralization assay (pCNA) was validated by comparison with a conventional CV-A16 neutralization assay (cCNA) in testing 174 human serum samples collected from children (age <5 years). The neutralizing antibody titers determined by these two assays were well correlated (R(2)=0.7689). These results suggest that the pCNA can serve as a rapid and objective procedure for the measurement of neutralizing antibodies against CV-A16.

Fig. 1

Generation of CV-A16 pseudoviruses. 293T cells were triple-transfected with the indicated plasmids to produce CV-A16 pseudoviruses containing luciferase. The pBS-T7-CV-A16-luc plasmid contains the reverse-transcribed full-length CV-A16 genome with the firefly luciferase gene in place of the P1 region. The VR1012-5′ NTR-P1 plasmid expresses the CV-A16 P1 gene. The pcDNA3.1-T7 RNA Pol plasmid expresses T7 RNA polymerase under control of the strong human CMV immediate early promoter. BGH: the bovine growth hormone polyadenylation (BGH-PolyA) signal, a specialized termination sequence for protein expression in eukaryotic cells.

Fig. 2

Characterization of CV-A16 pseudoviruses. (A) Western blot analysis of purified CV-A16 pseudoviruses using mouse anti-VP1 mAb. 1, marker; 2, non-transfected 293T cell lysates; 3, CV-A16-luc. The band corresponding to viral VP1 proteins (VP1) is indicated with an arrow. (B) Electrophoretic profile of PCR products of CV-A16 pseudoviruses. Lane 1, supernatants collected from cells transfected with pBS-T7-CV-A16-luc and pcDNA3.1-T7 RNA Pol; lane 2, supernatants collected from cells transfected with pBS-T7-CV-A16-luc, pcDNA3.1-T7 RNA Pol and VR1012-5′ NTR-P1; M, 1 kb DNA ladder (New England Biolabs: N3232). (C) Transmission electron microscopic observation of purified CV-A16 pseudoviruses. Bar, 50 nm.

Fig. 3

Determination of the optimal incubation time (A) and virus input (B–D) for pCNA. (A) Kinetics of CV-A16 pseudovirus infectivity in RD cells inoculated at MOI = 5 CCID₅₀ per cell. The cells were washed at 2 hpi, and then the luciferase activities in the cells were measured at the indicated times. Data are shown from one out of three experiments with similar results, each performed in quadruplicate. (B–D) Optimization of the CV-A16 pseudovirus titer for the pCNA. Neutralization curves of CV-A16 pseudoviruses (200, 400 and 800 CCID₅₀) with the standard anti-CV-A16 serum are shown. Error bars indicate standard deviations.

Fig. 4

Determination of threshold values for CV-A16 psedoviruses in pCNA for human sera. Distribution of neutralizing antibody titers in human serum samples (n = 174) determined by the pCNA with a range of threshold values. The results were compared with those obtained by the cCNA. The neutralizing antibody-positive rates of the samples were determined for each threshold value and are shown above the graphs. The threshold value of 20% showed neutralizing-antibody positive rates closest to those obtained by the cCNA (highlighted in red). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

Fig. 5

Correlation between CV-A16 neutralizing antibody titers from pCNA and cCNA. Neutralizing titers were measured by the cCNA and pCNA and log 2 transformed. The relative size of each spot corresponds to the number of the serum samples as indicated. R² = 0.7689.

Fig. 6

Distribution of neutralizing antibody titers measured by cCNA and pCNA in 174 serum samples.