Development of a pseudovirus-based assay for measuring neutralizing antibodies against Coxsackievirus A10

Abstract

Coxsackievirus A10 (CV-A10) has recently emerged as a major pathogen of hand, foot, and mouth disease in children worldwide. Currently no effective treatments are available; development of anti-CV-A10 vaccine is a most cost-effective way for CV-A10 prevention. Robust assay to measure neutralizing antibody (NtAb) titres elicited by vaccination would greatly prompt anti-CV-A10 vaccine development. Compare to the traditional neutralization assay based on inhibition of cytopathic effects (herein after referred to as cNT) which is time-consuming and labor-intensive, in this study we developed an efficient high-throughput neutralization antibody assay based on CV-A10 pseudoviruses (herein after referred to as pNT). In the pNT, anti-CV-A10 NtAb titre was negatively corresponded with the relative luminescent unit (RLU) produced by luciferase reporter gene incorporated in pseudovirus genome. As described in this study, the NtAb against CV-A10 could be detected within 10-16 h, anti- CV-A10 NtAb in 67 human serum samples were measured in parallel with pNT and cNT assays, a good correlation (r = 0.83,p < .0001) and good agreement(97%) were shown between cNT and pNT, indicating that the pNT provides a rapid and convenient procedure for measuring NtAb production against anti-CV-A10 NtAb measurement.

Keywords: Coxsackievirus A10; HFMD; cytopathic effect; neutralizing antibody; pseudovirus.

Figure 1.

Characterization of CV-A10 pseudovirus. (a) Development of the CV-A10 capsid expression construct, including the P1 gene and EGFP gene, with an EV-A71 2A protease cleavage site (AITTL) inserted upstream of the P1 sequence. (b) Electrophoretic profile of the PCR products of CV-A10 pseudovirus. The reporter gene Luc was detected in the supernatant of the HEK293T cell culture medium co-transfected with three plasmids. Lane 1: 1-kb ladder DNA marker; Lane 2: PCR products of the plasmid expressing Luc; Lanes 3 and 4: PCR products of the supernatant. (c) Immunoblot analysis for CV-A10 pseudovirus. VP1 protein was detected both in the CV-A10 pseudovirus and wild-type virus using mouse anti-VP1 mAb. Lane 1: protein marker; Lane 2: anti-VP1 mAb reacted with pseudovirus; Lane 3: anti-VP1 mAb reacted with wild-type virus. (d) CV-A10 pseudovirus neutralized by anti-CV-A10 serum based on detection of luciferase activity. (e) Specificity of CV-A10 pseudovirus tested against mouse anti EV-A71, CV-A16, CV-A6, CV-B3, and EV-D68 serum with the same amount of a diluted CV-A10 pseudovirus particle suspension (50 μl).

Figure 2.

Optimization of the CV-A10 pseudovirus luciferase assay. (a) Infectivity of CV-A10 pseudovirus in different susceptible cell lines. (b) Determination of optimal RD cell density. (c) Reaction time optimization. (d) CV-A10 pseudovirus infection linearity. Serially diluted CV-A10 pseudovirus was mixed with RD cells, and luciferase activity was measured at 10 h post-0infection. Points beyond the linear range were excluded.

Figure 3.

ROC curve analysis to estimate the degree of agreement between the cNT and pNT and to determine the cutoff value of the CV-A10 pseudovirus pNT. The hollow dot represents the cut off of CV-A10 pseudovirus.

Figure 4.

Correlation between the results of the CV-A10 pNT and cNT test. (a) Titres of anti-CV-A10 NtAb in human serum samples detected by the pNT and cNT analyzed by Spearman correlation (r = 0.83, P < .0001). (b) Titres of anti-CV-A10 NTAbs in human serum with the pNT and cNT compared by the Bland-Altman method.