Cross-Reactive Antibody Responses against Nonpoliovirus Enteroviruses

Figures

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  FIG 1. Binding of multiple isolates of EV-D68 by polyclonal sera from the sixth bleed of mice immunized with poliovirus type 1/Mahoney. Two-fold serial dilutions of polyclonal sera from mice inoculated with the poliovirus were incubated with 105 PFU of multiple isolates of EV-D68 that were either directly bound to the plastic surface (A) or captured by a recombinant human monoclonal antibody specific for EV-D68 (B) (45) overnight at 4°C, and binding was assessed by enzyme-linked immunosorbent assay (ELISA) and reported as the difference between excitation (470 nm) and emission (520 nm). The results are representative of three independent experiments.

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  FIG 2. Binding of poliovirus type 1/Mahoney by polyclonal sera from mice immunized with EV-D68, EV-D94, or EV-D111. Two-fold serial dilutions of monospecific polyclonal sera from mice inoculated with the EV-D68, EV-D94, or EV-D111 were incubated with 105 PFU of poliovirus type 1/Mahoney that was either directly bound to the plastic surface (A) or captured by anti-poliovirus type 1/Mahoney polyclonal rabbit sera (B) or human monoclonal antibody A12 that binds all polioviruses (C) (45) overnight at 4°C, and binding was assessed by ELISA and reported as the difference between excitation (470 nm) and emission (520 nm). The results are representative of three independent experiments.

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  FIG 3. Binding of representative EVs by guinea pig polyclonal sera from animals immunized with poliovirus type 1/Mahoney. Two-fold serial dilutions of polyclonal sera from guinea pigs immunized with poliovirus type 1/Mahoney were incubated with 105 PFU of NPEVs representing all species A to D and rhinoviruses. After incubation for 1 h at room temperature, binding was assessed by ELISA and reported as the difference between excitation (470 nm) and emission (520 nm). The results are representative of three independent experiments.

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  FIG 4. Inhibition of poliovirus type 1/Mahoney infection of murine fibroblasts producing the human poliovirus receptor by monospecific polyclonal anti-EV-D68 murine sera. Polyclonal sera were pooled from five wild-type mice immunized with an EV-D68 isolate from the 2018 outbreak that associated with AFM adjuvanted in Freund’s complete. Anti-EV-D68 or anti-poliovirus type 1/Mahoney monospecific polyclonal murine sera were diluted 1:1,024, incubated with 103 PFU of poliovirus type 1/Mahoney 1 h at room temperature, and overlaid onto a monolayer of murine fibroblasts producing the human poliovirus receptor. Cells and supernatant were collected at 0, 24, and 48 h postinfection, and neutralization of infectivity was assessed by plaque reduction assay. The results are representative of three independent experiments. PBS, phosphate-buffered saline.

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  FIG 5. Phylogenetic analysis of the capsid protein precursor (VP1 to VP4) of enteroviruses. Total collection includes 35 isolates of EV-D68, 3 human rhinoviruses (HRVs), and 6 enteroviruses of other species from EV-A, EV-B, EV-C, and EV-D. Enterovirus D-68 samples are further classified by clades A, B, and C. Phylogenetic analysis was conducted using a maximum likelihood model after applying ClustalW sequence alignment to all genome polypeptides with the MegaX program. Viruses highlighted in blue were found to be viruses that bound to anti-poliovirus type 1/Mahoney polyclonal sera. No binding was observed with the viruses highlighted in yellow.