Analysis of cross-reactive neutralizing antibodies in human HFMD serum with an EV71 pseudovirus-based assay

Huafei Zhang, Dong An, Wei Liu, Qunying Mao, Jun Jin, Lin Xu, Shiyang Sun, Liping Jiang, Xiaojun Li, Jie Shao, Hongxia Ma, Xueyong Huang, Shijie Guo, Haiying Chen, Tong Cheng, Lisheng Yang, Weiheng Su, Wei Kong, Zhenglun Liang, Chunlai Jiang, Huafei Zhang, Dong An, Wei Liu, Qunying Mao, Jun Jin, Lin Xu, Shiyang Sun, Liping Jiang, Xiaojun Li, Jie Shao, Hongxia Ma, Xueyong Huang, Shijie Guo, Haiying Chen, Tong Cheng, Lisheng Yang, Weiheng Su, Wei Kong, Zhenglun Liang, Chunlai Jiang

Abstract

Hand, foot and mouth disease, associated with enterovirus 71 (EV71) infections, has recently become an important public health issue throughout the world. Serum neutralizing antibodies are major indicators of EV71 infection and protective immunity. However, the potential for cross-reactivity of neutralizing antibodies for different EV71 genotypes and subgenotypes is unclear. Here we measured the cross-reactive neutralizing antibody titers against EV71 of different genotypes or subgenotypes in sera collected from EV71-infected children and vaccine-inoculated children in a phase III clinical trial (ClinicalTrials.gov Identifier: NCT01636245) using a new pseudovirus-based neutralization assay. Antibodies induced by EV71-C4a were cross-reactive for different EV71 genotypes, demonstrating that C4a is a good candidate strain for an EV71 vaccine. Our study also demonstrated that this new assay is practical for analyses of clinical samples from epidemiological and vaccine studies.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Phylogenetic tree of EV71 subgenotypes.
Figure 1. Phylogenetic tree of EV71 subgenotypes.
The phylogenetic tree was constructed by the neighbor-joining method with 1000 bootstraps based on nucleotide sequences of EV71 VP1 (891 bp) obtained from GenBank and maximum composite likelihood as the model. EV71-A (BrCr) was used as the outlier. The scale represents differences between sequences or the evolutionary distances used to infer the phylogenetic tree. The bootstrap value was indicated. These EV71 P1 genes were used to construct the plasmids that were prepared to generate the pseudoviruses.
Figure 2. Heatmap analysis of NtAb titers…
Figure 2. Heatmap analysis of NtAb titers of Changchun and Luoyang samples against EV71 pseudoviruses of different subgenotypes measured in a neutralization assay.
Thirteen positive serum samples were identified from the Changchun (CC) and Luoyang (LY) groups. The NtAb titers are shown in yellow for low, orange for middle and red for high titers. The heatmap was generated by a web tool (http://www.hiv.lanl.gov/content/sequence/HEATMAP/heatmap.html). NtAb titers against EV71 pseudoviruses of different subgenotypes, showing the cross-reactivity of the patients' sera, are indicated in the heatmap.
Figure 3. Heatmap analysis of serum antibody…
Figure 3. Heatmap analysis of serum antibody titers against EV71 pseudoviruses of different subgenotypes in vaccine-inoculated serum samples measured in a neutralization assay.
Thirty samples were divided into three groups according to the relative level of NtAb titer: low (L), middle (M) and high (H). The NtAb titers are shown in yellow for low, orange for middle and red for high titers. The web tool used to generate the heatmap is the same as that used for Figure 2. NtAb titers against EV71 pseudoviruses of different subgenotypes, showing the cross-reactivity of vaccine-inoculated sera, are illustrated in the heatmap.
Figure 4. Correlation analyses of NtAb titers…
Figure 4. Correlation analyses of NtAb titers against B4, B5, C2, C4a and C5 determined with CPE and pseudovirus assays.
The dots represent NtAb titers determined with the CPE and pseudovirus assays. Dotted lines represent the 95% confidence band, and solid lines were fitted from the NtAb titers measured by the two methods. NtAb titers against B4, B5, C2, C4a and C5, quantified with the pseudovirus assay, showed a statistically significant correlation with those detected with the CPE assay (Pearson's r≥0.6150, P<0.0001 in all cases).

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